Identification of modulators of neurotransmitter activity of xanthurenic acid

ABSTRACT

The invention relates to methods and compositions for the selection and development of novel pharmacological agents or novel medicaments having properties or mechanisms of action which are original. The invention specifically describes the characterization and identification of the functional role of several constitutive elements of the central nervous system playing a role in the metabolism or transport of xanthurenic acid (XA) or involved in receiving and/or transducing the signal mediated by this substance. Said targets are new tools for the definition and selection of novel synthetic chemical substances, interfering with said targets and modulating the functions of XA in order to benefit from the pharmacological or medicamentous advantages thereof. The invention can be primarily used to treat neurological or mental pathologies or diseases.

This application is the US national phase of international applicationPCT/FR02/02337 filed 4 Jul. 2002 which designated the US and claimsbenefit of FR 0108937, dated 5 Jul. 2001, the entire contents of each ofwhich is hereby incorporated by reference.

The invention relates to methods and compositions for the selection anddevelopment of novel pharmacological agents or novel medicaments havingproperties or mechanisms of action which are original. The inventionspecifically describes the characterization and identification of thefunctional role of several constitutive elements of the central nervoussystem playing a role in the metabolism or transport of xanthurenic acid(XA) or involved in receiving and/or transducing the signal mediated bythis substance. Said targets are new tools for the definition andselection of novel synthetic chemical substances, interfering with saidtargets and modulating the functions of XA in order to benefit from thepharmacological or medicamentous advantages thereof. The invention canbe primarily used to treat neurological or mental pathologies ordiseases.

The pharmaceutical industry is continually in search of new naturalsubstances, present in the brain, and which have an important functionalrole in the regulation and control of psychological, psychoaffective,mental, cognitive or neurological activity. The discovery of newmechanisms of neurotransmission and novel targets participating in saidmechanisms within the nervous system are just so many valuable tools forthe selection of novel medicaments in diverse indications to bedescribed hereinbelow. The present invention describes for the firsttime the important role of a natural substance, xanthurenic acid, incentral neurotransmission phenomena. This new neurotransmission systemcomprises a metabolic system for synthesis and elimination of thetransmitter, transporters, receptors and intracellular signals switchedon upon stimulation of said receptors. Such components constitute“targets” which may be modulated by potential medicaments and used forthe identification, selection or characterization of biologically activecompounds. Moreover, the invention also describes different functionalroles of xanthurenic acid in the brain (interference with otherneurotransmitters, role in some behavioral or neuropharmacologicalprotocols) allowing to envision novel pharmacological or therapeuticuses for said molecule, its derivatives or, more generally, natural orsynthetic substances which bind to the hereinabove targets.

Since the early 1980s, tryptophan catabolism has gradually assumed anever more prominent role in research on the causes and treatments ofmajor central nervous system disorders (Orv. Hetil. 1992, July 19; 133(29): 1803–1807). It is mainly compounds such as quinolinic acid andkynurenic acid, which are tryptophan metabolites, that have been thefocus of attention by virtue of their pharmacological profiles. Animbalance in the formation of these metabolites, a chronically alteredsynthesis or degradation, may underlie functional disturbances of thecentral nervous system and the resultant pathologies.

In the case of XA, another tryptophan metabolite, different studies havereported various effects of XA. For instance, XA (50–600 mg/kg i.p.) wasdescribed as having an analgesic effect in Sprague-Dawley rats in thehotplate (“plaque chaude”) and tail flick tests (Pharmacol. Res. 1998,October; 38 (4): 243–250). Moreover, in animals, intraperitonealinjection of XA prolonged the latency time to onset of epilepticseizures induced by intracerebroventricular injection of quinolinicacid, strychnine or pentylenetetrazole. It would seem that increasedexcretion of XA fulfils a compensation process in patients manifestingdifferent stages of convulsive episodes (J. Neurol. Transm. 1983; 56(2–3): 177–185; Clin. Endocrinol. (Oxf) 1997, December; 47 (6):667–677). After an oral dose of tryptophan (5 g), 24-hour urinaryexcretion of XA was markedly increased in depressed patients(Psychopharmacology (Berl) 1981; 75 (4): 346–349; Acta Psychiatr. Belg.1979, November–December; 79 (6): 638–646). Other results show howeverthat ICV injection of XA does not modify the ECG in rats (OkayamaIgakkai Zasshi 104 (5–6): 471–482, 1992), that injection of XA in micedoes not induce head twitch behavior (Psychopharmacology (Berl) 1977,March 16; 51 (3): 305–309), that XA does not block depolarizationsinduced by excitatory amino acids (Brain Res. 1986; August 20; 380 (2):297–302) and that XA induces an increase in the expression of anendoplasmic reticulum chaperone protein (Grp 94) and of calreticulin,causing an abnormal conformation of some proteins. The xanthurenic acidconcentrations required to produce these effects in cultured cells arein the millimolar range (H. Malina; Biochem. Biophys. Res. Comm., 1999,265: 600–605).

However, to date, the role of XA has yet to be characterized, its modeof action and its physiological role are still obscure, the possibleexistence of a receptor has not been demonstrated, and no therapeuticstrategy based on this molecule has been undertaken.

The present invention results from the description, characterization andidentification of the functional role of several constitutive elementsof the central nervous system, playing a role in the metabolism ortransport of xanthurenic acid (XA) or involved in receiving and/ortransducing the signal mediated by this substance. These targets are newtools for the definition and selection of novel synthetic chemicalsubstances, interfering with said targets and modulating the functionsof XA in order to benefit from the pharmacological and medicamentousadvantages thereof. The invention more particularly demonstrates therole of XA in the central nervous system and its involvement, as aneurotransmitter, in many pathophysiological mechanisms. The inventiondemonstrates in particular:

-   -   the existence and characteristics of extracellular release of XA        in the cortex,    -   the existence of a high affinity binding site for this substance        in brain, modulated by adenylate derivatives (adenosine, ADP,        ATP), copper and zinc ions, and kynurenic acid and its        derivatives,    -   the existence of functional interrelationships in vivo between        XA and dopamine in the brain, allowing in particular to propose        a role for XA nerve terminals in the frontal cortex in        regulating the dopaminergic system, in particular via an impact        on glutamatergic and GABAergic activities. XA induces        dose-dependent dopaminergic stereotypies when administered in        the A₁₀ nucleus, or in the nucleus accumbens, striatum and        lateral ventricles. This points to the XA system as an important        target for ligands of the corresponding receptor sites, said        ligands regulating glutamate/GABA/dopamine activites involved in        the production of schizophrenic symptoms. It might therefore be        expected that XA receptor ligands will be of major interest for        the treatment of certain psychotic symptoms,    -   induction of electrophysiological effects by micromolar        concentrations of XA on certain membrane ion conductances in        cells expressing XA receptors,    -   demonstration of sodium-dependent active transport of XA in        cultured neurons. This function represents a novel target for        structural analogs of XA, or more generally for synthetic        ligands interfering with said transport system. In fact,        inhibition of this transport system can result in an increased        half-life of XA in certain functional compartments in brain,        particularly the synaptic compartment. Inhibitors of this        transport system may therefore represent a novel class of        substances having beneficial effects in several types of        neurological or mental pathologies,    -   reactions to an acute stress lead not only to extracellular        release of norepinephrine, but also to a marked release of XA.        Release of the latter substance participates in producing        adaptation reactions to said stress. The results therefore        indicate that ligands modulating the activity of the XA system        may be useful in man for regulating reactions to stress.    -   the ability of synthetic XA agonists to induce XA-type        electrophysiological effects in NCB-20 cells and, in particular,        to modulate cerebral dopamine in vivo,    -   the ability of XA antagonists, such as NCS-486, to antagonize        the electrophysiological effects induced by XA or its agonists,        and in particular to inhibit the dopaminergic activity of XA in        vivo, and    -   the specific neuropharmacological effects of XA in vivo, after        local application or systemic administration, which induces        sedation, anxiolysis, dopaminergic effects (which may be useful        in several areas such as drug addiction, Parkinson's disease,        schizophrenia, mood disorders, and the like), antidepressant        effects and beneficial effects on memory and social        interactions.

The present application thereby demonstrates that XA, for which nofunctional role had so far been identified in the CNS (central nervoussystem), has properties that strongly suggest a role as aneurotransmitter/neuromodulator. This newneurotransmission/neuromodulation system offers an array of potentialtargets (enzymes that synthesize and degrade XA, receptor sites,transport sites, sites of release) for ligands or compounds, ofsynthetic or natural origin, modulating the activity of XA and, for thisreason, representing potential medicaments in the therapy ofneurological and mental disorders.

The present application therefore has as its object the use of acompound modulating the activity of xanthurenic acid for preparing amedicament for treating nervous system pathologies. The invention alsoconcerns a method for treating pathologies of the nervous system,particularly central, comprising administering to a subject a compoundmodulating the activity of xanthurenic acid.

A particular aspect of the invention is based on the use a compoundspecifically modulating xanthurenic acid activity for preparing amedicament for treating nervous system pathologies, and on acorresponding method of treatment. The compound is called specific whenit modulates foremost the activity of XA [e.g., its transport, itsmetabolism or the activity of XA receptor(s)] without being primarilyintended to have a significant, direct effect on the activity of othercentral neurotransmission/neuromodulation systems. Of course, modulationof the central XA system leads to consequences and adaptations in othersystems, in particular the dopaminergic or GABAergic system, but notsubstantially in the serotoninergic system. Particular specificcompounds according to the invention are compounds able topreferentially bind to an XA receptor without binding, in a specific orsubstantial manner, to a serotoninergic receptor or other receptor ofother neurotransmitters/neuromodulators.

According to preferred modes of embodiment, the invention has moreparticularly as its object:

-   -   the use of a compound modulating xanthurenic acid activity for        preparing a medicament for treating anxiety, and a corresponding        method of treatment;    -   the use of a compound modulating xanthurenic acid activity for        preparing a medicament for treating depression, and a        corresponding method of treatment;    -   the use of a compound modulating xanthurenic acid activity for        preparing a medicament for treating impairment of memory or        social interactions, and a corresponding method of treatment;    -   the use of a compound modulating xanthurenic acid activity for        preparing a sedative and/or hypnotic medicament, in particular a        compound which potentiates the neuropharmacological effect of        XA, preferably a compound which is an allosteric activator of        the XA receptor, and a corresponding method of treatment;    -   the use of a compound modulating xanthurenic acid activity for        preparing a medicament for modulating dopaminergic action, and a        corresponding method of treatment;    -   the use of a compound modulating xanthurenic acid activity for        preparing a medicament for treating psychotic symptoms,        particularly schizophrenia, and a corresponding method of        treatment;    -   the use of a compound modulating xanthurenic acid activity for        preparing a medicament for regulating reactions to stress, and a        corresponding method of treatment;    -   the use of a compound modulating xanthurenic acid activity for        preparing a medicament for regulating behavioral arousal, and a        corresponding method of treatment;

Within the context of the invention, the term “treatment” denotespreventive, curative, palliative treatment as well as management ofpatients (alleviating suffering, prolonging survival, improving qualityof life, slowing disease progression), etc. Furthermore, the treatmentmay be carried out in combination with other agents or treatments,particularly addressing the late events of the disease, or with otheractive substances. As noted hereinabove, the compounds used arepreferably specific modulators of XA activity.

The invention also has as its object a method for modulating thedopaminergic effects of XA comprising administering to a subject acompound modulating the activity of xanthurenic acid.

A further object of the invention is the use of a compound modulatingxanthurenic acid activity for preparing a medicament for treatingneurodegenerative diseases, particularly Parkinson's disease,Alzheimer's disease or ALS, mental illnesses, such as schizophrenia, orelse some drug dependencies, particularly opioid.

The invention equally concerns a method for modulating XA activity invivo, comprising administering to a subject an efficacious dose of asynthetic compound modulating XA activity.

In the context of this application, the term “modulation” denotes eithera stimulation, or an inhibition of XA activity. Stimulation may beachieved by using compounds mimicking the activity of XA or stimulatingthe quantity or efficacy of XA. Inhibition may be partial (eg., areduction of activity). It may be achieved by decreasing the amount ofXA present or by inhibiting the ability of XA to produce a biologicaleffect, or by blocking or inhibiting the biological effect induced byXA.

For this reason, in the context of the present application, the term“xanthurenic acid activity” denotes in particular the synthesis of thiscompound, its transport, its release, its interaction with a receptor orwith a partner, its degradation, a signal or metabolic pathway activatedor regulated by XA, and the like, that is to say, the activity of thesystem using XA as mediator. The compound modulating XA activity maytherefore be an agent that modulates XA synthesis, an agent modulatingXA transport or release, an XA agonist, an XA antagonist, and the like.It may also be a compound that modulates the activity of the XA receptorsite by acting on a regulatory or allosteric site of said receptor.

According to a first specific embodiment, the compound used is acompound modulating the synthesis or regulation of XA. Such compound mayact on the activity or expression of enzymes involved in thebiosynthesis of this molecule or its precursors, in particular acompound modulating the activity of kynurenin-3-hydroxylase. This enzymeplays a role in regulating XA production, and its modulation by thecompounds enables modulation of XA activity, within the context of thepresent application. Compounds modulating the activity or expression ofthis enzyme are for instance antisense molecules, transcriptionalinhibitors, ribozymes or aptazymes, anti-protein antibodies, chemical orpeptide compounds able to inhibit the activity of the enzyme, etc.

According to another particular embodiment, the compound used is acompound modulating the binding of XA to a membrane receptor (eg., anagonist or antagonist). An agonist is a compound displaying an affinityfor the XA binding site, and producing a signal analogous to thatproduced by XA. An antagonist is a compound displaying an affinity forthe XA binding site, and preventing binding and signal production by XAor an XA agonist. Such compounds may be XA analogs, anti-XA antibodies,synthetic compounds and the like. Thus, a compound modulating XA bindingto a membrane receptor implicates the XA system as an important targetfor ligands of the corresponding receptor sites, said ligands regulatingglutamate/GABA/dopamine activities involved in the production ofschizophrenic symptoms. XA receptor ligands are of particular interestfor treating certain psychotic symptoms. XA receptor antagonistcompounds find use more specifically in regulating behavioral arousaland in certain mental illnesses or in hyperdopaminergic states. Suchcompounds may be identified, synthesized, or characterized by themethods described hereinbelow.

Another usable compound according to the invention is a compoundmodulating XA transport or release. Such compounds, by reducing orfacilitating XA release, allow local modulation of XA activity, in thecontext of the invention. Other synthetic molecules, having in somecases structural analogies with XA, may interfere with its transport invesicles or via plasma membrane transporters. In particular, thedemonstration of a sodium-dependent active transport of XA in culturedneurons represents a novel target for XA structural analogs, or moregenerally for synthetic ligands interfering with said transport system.In fact, inhibition of this transport system results in an increase inthe half-life of XA in certain functional compartments of brain, inparticular the synaptic compartment. Inhibitors of this transportthereby represent a novel class of substances having beneficial effectsin several types of neurological or mental pathologies.

Another useful compound according to the invention is a compoundmodulating the synthesis, transport or activity of the XA receptor. Suchcompounds, by reducing or facilitating exposure of XA receptors, allowlocal modulation of the activity of this molecule.

Compounds modulating XA activity according to the invention may be ofdiverse nature and origin. They may be inorganic or organic products andnotably a polypeptide (or a protein or a peptide), a nucleic acid, alipid, a polysaccharide, a chemical or biological compound, and thelike. The compound may be of natural or synthetic origin and inparticular come from a combinatorial library. Preferably, such compoundsare derivatives of xanthurenic acid or kynurenic acid.

The compounds or compositions of the invention may be administered indifferent ways and in different forms. For instance, they may beadministered by the systemic or oral route, preferably systemically,such as for example by the intravenous, intramuscular, subcutaneous,transdermal, intra-arterial, intracerebral, intraperitoneal,intracerebrovascular route, etc. For injections, the compounds aregenerally prepared in the form of liquid suspensions, which may beinjected through syringes or by infusion, for instance. In this respect,the compounds are generally dissolved in pharmaceutically compatiblesaline, physiologic, isotonic, buffered solutions and the like, known tothose skilled in the art. For instance, the compositions may contain oneor more agents or vehicles chosen from among dispersives, solubilizers,stabilizers, preservatives, and the like. Agents or vehicles that may beused in the liquid and/or injectable formulations comprise in particularmethylcellulose, hydroxymethylcellulose, carboxymethylcellulose,polysorbate 80, mannitol, gelatin, lactose, vegetable oils, acacia, andthe like. The compounds may also be administered in the form of gels,oils, tablets, suppositories, powders, capsules, gelules and the like,possibly by means of pharmaceutical forms or devices allowing sustainedand/or delayed release. For this type of formulation, an agent such ascellulose, carbonates or starches is advantageously used.

It is understood that the injection rate and/or injected dose may beadapted by those skilled in the art according to the patient, thepathology, the mode of administration, etc. Typically, the compounds areadministered at doses ranging from 0.1 μg to 1000 mg/kg of body weight,more generally from 0.01 to 500 mg/kg, typically between 1 and 200mg/kg. Furthermore, repeated injections may be given, as the case maybe. In addition, in the case of chronic treatments, delayed or sustainedrelease systems may be advantageous.

As a rough guide, the results presented in the examples reveal that XAhas a sedative effect when administered to animals at doses greater thanapproximately 50 mg/kg. The preferred doses to obtain an anxiolyticeffect in animals are doses of less than 50 mg/kg. An antidepressanteffect is advantageously obtained in animals at doses comprised between100 and 200 mg/kg.

The invention also concerns methods to identify, select or characterizebiologically active compounds, particularly compounds active on thenervous system, said methods being based on modulation of XA activity.The inventive methods may comprise in vitro binding tests, binding testsin cell systems (or on natural or synthetic membrane preparations) orfunctional tests in cell or artificial systems.

According to a particular embodiment, a method for selecting,identifying or characterizing compounds according to the inventioncomprises contacting a test compound with a cell (or a natural orsynthetic membrane preparation) expressing a target molecule involved inxanthurenic acid activity, and demonstrating a binding of the testcompound to said target.

The target molecule may be the XA receptor, an XA synthetic enzyme, anXA transporter, etc., or any fragment thereof. In a specific example,the target molecule is the XA receptor or a fragment thereof,particularly a fragment conserving the ability to bind XA, such as forinstance an extracellular fragment. In another embodiment, the targetmolecule is an enzyme involved in XA biosynthesis or regulation, such asfor example kynurenin-3-hydroxylase. A preferred target molecule isrepresented by the XA receptor or fragments thereof.

The binding of the test compound to the target molecule may bedemonstrated for example by using a labelled test compound or by using alabelled ligand of the target molecule (for example a labelled XAreceptor ligand or a labelled antibody specific of an XA syntheticenzyme or an XA transporter), displacement of the binding of thelabelled ligand reflecting the binding of the test compound. Thelabelled ligand may be any product which binds the target molecule(antibody, agonist or antagonist, fragment or derivative of anendogenous ligand, etc.). The ligand may be labelled by any method knownto those skilled in the art, particularly by incorporation of aradioactive, fluorescent, luminescent, enzymatic, colorimetric moiety,etc.

In a particular embodiment, the ligand is a labelled antibody, specificof the target molecule under study. The antibody may be polyclonal ormonoclonal. It may also be an antibody derivative or fragment, such asfor example the Fab, Fab′2, CDR fragments, etc. The antibodies may beproduced by conventional means, by immunizing the target molecule (or animmunogenic part thereof) and recovering serum (polyclonal) or spleencells (for hybridoma production through fusion with an appropriateline), such as described for example in Vaitukaitis et al. (J. Clin.Endocrinol. Metab. 1971, 33(6): 988–91) or in Harlow et al. (Antibodies:A Laboratory Manual, CSH Press, 1988). The Fab or F(ab′)2 fragments maybe produced for example as described in Riechmann et al., 1988, Nature332, 323–327.

In another particular embodiment, the target molecule is the XA receptorand the ligand is an endogenous ligand, an agonist or an antagonist ofthe receptor, which is labelled. In a specific embodiment, labelled XA,particularly radiolabelled, in particular tritiated, is used.

According to a particular embodiment, the method therefore comprisescontacting a test compound with a cell (or a natural or syntheticmembrane preparation) expressing a xanthurenic acid receptor anddemonstrating a binding of the test compound to the receptor.

According to another particular embodiment, the method comprisescontacting a test compound with a cell (or a natural or syntheticmembrane preparation) expressing a target molecule involved inxanthurenic acid activity, in the presence of a labelled ligand of saidtarget, and demonstrating a binding of the test compound by measuringthe displacement of binding of the labelled ligand.

In a further variant, the method of the invention comprises contacting atest compound with a cell expressing a target molecule involved inxanthurenic acid activity (in particular the xanthurenic acid receptor),and demonstrating a biological or pharmacological effect characteristicof a binding of the test compound to said target molecule. Thebiological or pharmacological effect may be the expression of one ormore cellular proteins, the expression of a receptor, the activation ofa gene, the endocytosis of the receptor, the appearance of an electricalcurrent, a flux or influx of ions, etc. Such effect may be demonstratedby any suitable means, such as measuring the expression of a reportergene, measuring the membrane expression of a receptor, assaying ions orelectrical current, and the like.

Classically, the effect of the test compound is compared to the effectdetermined in the absence of said compound. Furthermore, the effect ofthe test compound may be determined in the presence of an XA receptorligand, for example XA itself, particularly when an XA modulating orinhibiting activity is sought.

According to a particular variant, the method of the invention thereforecomprises contacting a test compound with a cell expressing axanthurenic acid receptor, in the presence of a ligand of said receptor,measuring a biological or pharmacological effect characteristic of abinding to the XA receptor and comparing the effect so measured withthat obtained in the absence of the test compound. Such method isespecially suited to the research, selection, characterization orimprovement of compounds that inhibit XA activity.

The cells used in the tests may be any cells expressing a targetmolecule involved in XA activity, particularly an XA receptor. They maybe cells naturally expressing this molecule (e.g., this receptor), orcells genetically modified or treated so as to overexpress said molecule(e.g., said receptor). In a preferred embodiment of the invention, theyare mammalian cells (nerve cells, hepatocytes, fibroblasts, endothelial,muscle cells, etc.). Even more preferably, such cells are human cells.They may also be primary cell cultures or established cell lines. Inanother embodiment, it is also possible to use prokaryotic cells(bacteria), yeast cells (Saccharomyces, Kluyveromyces, etc.), plantcells, and the like.

In a preferred embodiment, nerve or synpatic cells are used,particularly neurons, glial cells, astrocytes, material of synapticorigin (membranes, synaptosomes, synaptoneurosomes), etc. Such cells maybe isolated, cultured and characterized according to known methods,described in the examples.

The invention demonstrates, for the first time, the existence of a highaffinity receptor for XA expressed in different cell populations orregions of the brain. Said receptor has the following pharmacologicalfeatures:

-   -   expression in brain,    -   K_(d) of the synaptic XA receptor less than or equal to        approximately 300 nM to 1300 nM,    -   receptor modulated by adenylate derivatives (adenosine, ADP,        ATP)    -   receptor modulated by copper and zinc ions,    -   receptor modulated by kynurenic acid,    -   receptor activation by XA induces an electrical current,    -   receptor expressed by NCB-20 cells.

Moreover, electrophysiological and biochemical data suggest a receptorcoupled to G proteins, although this has not been formally demonstrated.

The characterization of this receptor, its identification and thedescription of the methods whereby to measure the binding of moleculesto said receptor, according to this application, make it possible todemonstrate novel biologically active compounds, capable of modulatingXA activity. The demonstration of the pharmacological role of XAemphasizes the importance of making such active compounds available.

In a particular embodiment, the hereinabove methods are carried out byusing a membrane preparation expressing an XA receptor. In anadvantageous manner, the membrane preparation is of natural origin, thatis to say, produced from a cell expressing the receptor. Membranepreparations may be produced by mechanical, chemical, physical,electrical lysis, etc. and in particular by treatment with detergents,ultrasound, freeze/thaw, etc., as illustrated in the examples. Suchmembrane preparation is characterized primarily by the presence ofmembrane fragments, containing a lipid bilayer in which all or part ofthe XA receptor is present. Such membrane preparations are generallydevoid of intact cells. Furthermore, they may be enriched in membranedebris by suitable treatments (centrifugation, etc.). A membranepreparation of synthetic origin may also be used, such as for example aliposome in which all or part of the XA receptor has been inserted, or asupported membrane.

For the binding or functional tests hereinabove, the compounds may becontacted with the cells (or membrane preparations) at different times,depending on their effect(s), their concentration or the type of celland for different periods, which may be adapted by those skilled in theart. The test may be carried out on any suitable support andparticularly on a plate, slide, dish, in a tube or flask. Generally thecontact is done in a multiwell plate which makes it possible tosimultaneously conduct numerous and diverse tests. Typical supportsinclude microtitration plates and more particularly plates with 96 or384 wells (or more), which are easy to manipulate.

Depending upon the support and the nature of the test compound, variableamounts of cells may be used when carrying out the described methods.Classically, 10² to 10⁶ cells are contacted with a type of testcompound, in a suitable culture medium, and preferably between 10³ and10⁵ cells. When a membrane preparation is used, 0.01 to 50 mg perprotein per test is generally used, more preferably from 0.05 to 2 mg ofprotein per test. The tests may be carried out in any suitable medium,such as for example saline solutions, buffers, etc. Specific examples ofbuffers include Tris, Pipes, Hepes, etc. The temperature is typicallyclose to room temperature. The pH of the medium is advantageouslycomprised between 5.5 and 8, more preferably between 7 and 8. It isunderstood that these parameters may be adapted by those skilled in theart, following the indications given in the examples.

The quantity (or concentration) of test compound may also be adjusted bythe user according to the type of compound (its toxicity, ability topenetrate cells, etc.), the incubation time, etc. Generally, the cells(or membranes) are exposed to quantities of test compound ranging from 1nM to 1 mM. Of course other concentrations may be tested withoutdeviating from the invention. Each compound may, furthermore, be testedin parallel, at different concentrations and for different times.Moreover, adjuvants and/or vectors and/or products facilitatingpenetration of the compounds into the cells such as liposomes, cationiclipids, polymers, viral peptides, etc. may additionally be used, wherenecessary. Contact may be maintained for a period comprised between 1minute and several hours, according to the nature of the targetmolecule. When the target molecule is the XA receptor, contact istypically for less than approximately 1 hour. When the target moleculeis an intracellular molecule, contact may be maintained for a longertime.

In another variant, the invention is based on in vitro binding testsbetween XA and a test compound or between a test compound and a targetmolecule (for example all or part of the XA receptor). According to suchembodiments, the method by which to select, identify or characterizeactive compounds comprises:

-   -   contacting a test compound with a target molecule involved in XA        activity, for example a synthesis or regulatory enzyme, a        transporter, a receptor, or a fragment thereof, and    -   determining the possible binding of said test compound to said        molecule.

Binding of the test compound may be demonstrated in different ways, suchas for example by gel migration or electrophoresis of the complexesformed. Other methods based on luminescence or the FRET (FluorescenceResonance Energy Transfer) method familiar to those skilled in the artor the SPA (Scintillation Proximity Assay) method, may be used, withinthe scope of the present invention, to determine the possible binding ofthe test compound to the target molecule. When the target is a receptor,binding may be measured in the presence of a labelled ligand, bymeasuring displacement of the ligand by the test compound.

The present invention may be applied to any type of test compound. Forinstance, the test compound may be any product alone or mixed with otherproducts. The compound may be defined in terms of its structure and/orcomposition or it may not be defined. The compound may, for example, bean isolated and structurally defined product, an isolated product ofundefined structure, a mixture of known and characterized products or anundefined composition comprising one or more products. For example, suchundefined compositions may be samples of tissue, biological fluids,cellular supernatants, plant preparations, etc. The test compounds maybe inorganic or organic products and particularly a polypeptide (or aprotein or a peptide), a nucleic acid, a lipid, a polysaccharide, achemical or biological compound such as a nuclear factor, a cofactor orany mixture or derivative thereof. The compound may be natural orsynthetic and include a combinatorial library, a clone or a library ofnucleic acid clones expressing one or more DNA-binding polypeptide(s),etc.

Another object of the invention relates to a method for producing anactive compound, particularly on the nervous system, the methodcomprising:

-   -   determining the ability of a compound to modulate XA activity in        vitro or ex vivo, and    -   synthesizing said compound or a structural analog thereof.

Another object of the invention concerns a method for producing amodulator of XA, the method comprising:

-   -   contacting a compound with a target molecule involved in XA        activation,    -   determining the effect of the compound on the target molecule,        said effect indicating that the compound is a modulator of XA,        and    -   synthesizing said compound or a structural analog thereof.

A further object of the invention concerns a method for producing amedicament comprising an active compound, particularly on the nervoussystem, the method comprising:

-   -   determining the ability of a compound to modulate XA activity in        vitro or ex vivo, and    -   combining said compound or a structural analog thereof with a        pharmaceutically acceptable vehicle.

Another object of the invention concerns a method for producing amedicament comprising an XA modulator compound, the method comprising:

-   -   contacting a compound with a target molecule involved in XA        activation,    -   determining an effect of the compound on the target molecule,        said effect indicating that the compound is a modulator of XA,        and    -   combining said compound or a structural analog thereof with a        pharmaceutically acceptable vehicle.

Within the scope of the invention, the term “structural analog” denotesany molecule obtained by molecular modelling or structural variationfrom a test compound.

Other advantages and aspects of the invention will become apparent inthe following examples, which are given for purposes of illustration andnot by way of limitation.

LEGENDS OF FIGURES

FIG. 1: Extracellular concentration of XA in the prefrontal cortex (PFC)

FIGS. 2A and 2B: XA release in the PFC after electrical stimulation ofthe VrA (FIG. 2A=100 μA; FIG. 2B=200 μA). Dopamine=black triangles;XA=black squares

FIG. 3: XA release induced by 100 mM KCl in the probe for 5 min.

FIG. 4: XA release induced by 50 μM veratridine in the probe for 20minutes.

FIG. 5: XA release in the PFC after electrical stimulation (white bars).The same experiment in the absence of calcium ions and in the presenceof EGTA shows that XA release is no longer increased after electricalstimulation (black bars).

FIG. 6: XA release in the PFC after electrical stimulation of the VTA(white bars, baseline=21 pmol/5 min). Same experiment but with 2.0 μMTTX in the dialysis medium (black bars, baseline=7 pmol/5 min). In thepresence of TTX, electrically-induced XA release is blocked.

FIG. 7: Distribution of XA in rat brain under physiological conditions.Concentrations are arbitrarily color coded. Abbreviations: PFC:prefrontal cortex; FC: frontal cortex; PC: parietal cortex; OC:occipital cortex; C: cerebellum; GP: globus pallidus; CPu: caudateputamen; n.Acc: nucleus accumbens; OT: olfactive tubercules; OB:olfactive bulbs; S: septum; Hb: habenula; Hi: hippocampus; Th: thalamus;Hy: hypothalamus; SN: substantia nigra (A9); VTA: ventral tegmental area(A10); Rad: dorsal raphe nucleus; Ram: median raphe nucleus; LC: locuscaeruleus (A6); MO: medulla oblongata.

FIG. 8: Effect of pH on XA binding to its binding site(s).

FIG. 9: Linearity study of specific binding according to proteinconcentration.

FIG. 10: Determination of association constant K_(on)

FIG. 11: Determination of dissociation constant K_(off)

FIG. 12: Saturation curve of XA membrane sites. Determination of K_(d)and B_(max).

FIG. 13: Competitive inhibition of radiolabelled XA binding bynon-radiolabelled XA. Determination of IC₅₀ for xanthurenic acid.

FIG. 14: Effect of metal ions on xanthurenic acid binding to its bindingsite(s).

FIG. 15: Effect of pH on XA binding to its binding site(s) in thepresence of copper ions.

FIG. 16: Linearity study of specific binding according to proteinconcentration in the presence of copper ions.

FIG. 17: Dose-effect of copper (Cu²⁺) on XA binding to its bindingsite(s).

FIG. 18: Saturation curve and K_(d) and B_(max) determination in thepresence of copper ions.

FIG. 19: Competitive inhibition of radiolabelled XA binding bynon-radiolabelled XA. Determination of IC₅₀ for xanthurenic acid.

FIG. 20: Dose/effect of XA on the dopaminergic response after localinjection in the PFC.

FIG. 21: Variations in dopamine, DOPAC and HVA release in the PFC afterlocal application of 20 μM XA.

FIG. 22: Effects of the antagonist NCS-486 on extracellular dopaminerelease after local application of 20 μM XA in the PFC.

FIG. 23: Experimental configuration of patch-clamp studies.

FIG. 24: Effect of xanthurenic acid on NCB-20 cells.

-   -   A. Sample traces recorded on a membrane fragment under control        conditions and during application of 20 μM xanthurenic acid. The        membrane fragment was stimulated by a potential ramp as        indicated in the protocol shown in the lower trace. In the        presence of xanthurenic acid a large outflux current develops at        positive potentials. The trace was digitized at a frequency of 2        kHz.    -   B. Variations in the mean outflux current measured at a        potential of 95 mV. In the presence of xanthurenic acid this        current is considerably increased.    -   C. Current-potential relationship of the active current in the        presence of xanthurenic acid (after subtracting the current        observed in control condition). The solid line fits this current        to the Boltzmann model.

FIG. 25. Pharmacology of the response to xanthurenic acid. The membranecurrent measured at potentials of −80 and 80 mV is plotted againstrecording time. Solid lines indicate the incubation periods of thexanthurenate receptor ligands: upper and lower respectively for NCS-482and NCS-486 alone and middle for the mixture of the two at the indicatedconcentrations. NCS-482 and NCS-486 are xanthurenic acid derivatives.NCS-486 alone does not change the current amplitude.

FIG. 26: Activation of chloride conductance by the xanthurenic acidreceptor. The agonist used in this experiment is NCS-482 at 20 μMconcentration. The response develops in two phases (a and b). The natureof the current in these two phases differs as shown by the inversionpotential of the current obtained in each of the two phases (lowerpanels).

FIG. 27: Spontaneous locomotor activity of animals treated withincreasing doses of XA. Time is given in minutes.

FIG. 28: Global reactivity of animals in the open field test. XA at adose of 37.5 mg/kg (non-sedative dose) promotes movement of the animalto the center of the lighted open field as compared to control animals.

FIG. 29: Dose/effect of XA in the Porsolt test (swimming test). Theduration of immobility decreases with increasing doses of XA. Ratstreated with imipramine 30 mg/kg served as positive controls.

FIG. 30: In the open field test, an XA dose of 50 mg/kg induces anincrease in the duration of social interactions compared to untreatedanimals. This result also suggests that XA has an anxiolytic effect.

FIG. 31: Effect of XA on memorization processes in the objectrecognition test.

FIG. 32: Transport kinetics of XA (ND-1089) in NCB-20 cells. The maximaltransport velocity is reached after approximately 1 minute.

FIG. 33: XA (ND-1089) transport in NCB-20 cells according to [³H]-XAconcentration. The Scatchard plot allows determination of themathematical constants of intracellular transport of ND-1089: K_(m)=105μM and V_(max)=1229 pmol/mg protein/minute.

FIG. 34: Histogram showing the percentage uptake relative to transportin the presence of radiolabelled XA alone (=100%).

FIG. 35: Frontal extracellular release after retrodialysis of 20 μM XAfor 20 minutes expressed as dopamine concentration in the dialysates (%of baseline) versus time (minutes).

FIG. 36: Frontal extracellular release after retrodialysis of 20 μM XAfor 20 minutes expressed as glutamate concentration in the dialysates (%of baseline) versus time (minutes).

FIG. 37: Frontal extracellular release after retrodialysis of 20 μM XAfor 20 minutes expressed as glutamate concentration (% of baseline)versus time (minutes).

FIG. 38: Actograph obtained after administration of a xanthurenic acidderivative ligand named ND-7000, which binds to the allosteric site forkynurenic acid, an integral part of the XA receptor, at a dose of 100mg/kg per os.

FIG. 39: Dose/response after p.o. administration of an XA receptoragonist—a xanthurenic acid derivative named ND-1301—on dopamine tissuelevels in different parts of the brain.

EXPERIMENTAL SECTION 1. Demonstration and Characteristics ofExtracellular Release of Xanthurenic Acid (XA) In Vivo in Rat PrefrontalCortex.

Through a combination of electrical stimulation of the ventral tegmentalarea (A₁₀) and in vivo microdialysis in the prefrontal cortex in rats,extracellular release of XA could be demonstrated. This release can bereproduced by local depolarization induced by high potassium ionconcentrations or micromolar quantities of veratridine. XA release iscalcium-dependent and is blocked by tetrodotoxin which prevents localdepolarization of neurons. XA is therefore released in this region ofthe brain at least, with characteristics identical to those of otherknown neurotransmitters, that is to say, according to an exocytoticmechanism induced by neuron depolarization.

Materials and Methods

Animals

Male Wistar rats weighing 350–400 g were used for the experiments.Animals were housed individually in plastic cages with a light/darkcycle of 7 a.m./7 p.m. and 7 p.m./7 a.m. Animals had access to food andwater ad libitum. Animal experiments were conducted in strict accordancewith the requirements of the European Directive of 24 Nov. 1986(86/609/EEC).

Surgical Procedure

The experiments were carried out using an L-shaped cannula with a 4 mmtip. A polycarbonate-polyether dialysis membrane (500 μm) with a 20 Kdacutoff was used. The dialysis membrane was inserted in the prefrontalcortex (PFC) while one of the tips of the bipolar stimulation electrodewas implanted in the PFC and the other in the VTA. The procedure wasperformed under ketamine anesthesia.

Microdialysis Protocol

The experiments were carried out in conscious animals, 24 to 72 hoursafter the surgery. The perfusion medium had the following composition:147 mM NaCl, 1.2 mM CaCl₂, 1.2 mM MgCl₂, and 4.0 mM KCl, pH 6.5. In thecase of stimulations at high potassium concentrations, sodium ionconcentrations were lowered to the same value. In the case ofstimulations in the absence of calcium ions, 2.0 mM EGTA was added tothe dialysis medium. Dialysates were collected at a rate of 2 μl/min,every 5 minutes, and immediately stored in liquid nitrogen up toanalysis.

Analysis of Dopamine and Xanthurenic Acid

These two compounds were quantitatively determined by HPLCchromatography with electrochemical detection. The chromatographicsystem consisted of a 25 cm×4.6 mm Hypersyl C18 column, maintained atconstant temperature. The mobile phase was 0.05 M NaH₂PO₄ buffer+0.1 mMEDTA and 6% methanol. Peaks were identified by comparison with theretention times measured on calibration standard solutions.

Electrical Stimulation Protocol

The VTA was stimulated for a period of 10 minutes, with bursts of thirty0.5-msec pulses for 100 millisec, intensity 100 or 200 μA and at afrequency of 25 Hz.

Determination of In Vitro Yields

These determinations were designed to estimate the quantity of dopamineand XA that crossed the dialysis membrane in the in vivo experiments.Dialysis yields in vitro were approximately 28% and approximately 15%for XA and dopamine, respectively, for a dialysis rate of 1 μl/min atroom temperature.

Results

Determination of XA Concentrations in the Extracellular Medium of thePFC

These studies were carried out by determining the differentconcentrations of XA dialyzed at different dialysis yields. The variousdialysis yields were modified by gradually changing the flow rate of thedialysis medium (1, 2, 3 and 4 μl/minute, respectively) and determiningthe XA concentration in each case. Extrapolation to a rate of zero givesa yield of 100% and an XA concentration in the dialysis medium equal toits concentration in the extracellular medium. The experiment, conductedon 3 different rats, gave a value of XA=6.1 pmol/μl of dialysis mediumor cerebral extracellular medium, which gives a mean value of 6.1 μM XAin the extracellular medium of the PFC (FIG. 1).

XA release in PFC After Electrical Stimulation

In two series of experiments, the electrical stimulation was 100 or 200μA for 10 minutes. After inserting the probe and dialyzing for 1 hour,the baseline value was calculated by dialyzing for a second hour(baseline: 16 to 18 pmol/5 min). A stimulation at 100 or 200 μA gave thesame result, indicating that the entire compartment that could bereleased in our conditions, was released. Compared to a baselinearbitrarily set to 100%, electrically-induced XA release reached 539%while dopamine release reached 655%. Release started immediately afterthe electrical stimulation, peaked after about 5 minutes of stimulation,then returned to baseline, even if stimulation was continued. A moreprecise kinetic profile could not be obtained, given the dialysisconditions (5-minute fractions). The results are shown in FIG. 2.

XA release in PFC After Local Depolarization Induced by HighConcentrations of Potassium Ions or by 50 μM Veratrine

XA release in the PFC extracellular medium was also measured after localdepolarization induced by high potassium ion concentrations in thedialysis probe (100 mM KCl for 5 minutes). Before stimulation, baselinerelease was approximately 92 pmol/20 min. After depolarization, XArelease gradually increased to reach 250% of the baseline value after 40minutes. Levels then gradually returned to baseline which was reached 80minutes after depolarization (FIG. 3). In the case of veratrine, a 50 μMconcentration was placed in the probe for 20 minutes. Baseline XArelease (before depolarization) was approximately 96 pmol/20 minutes.After application of veratrine, XA release peaked rapidly, reachingapproximately 600% of baseline after 20 minutes, then gradually returnedto baseline within about 60 minutes (FIG. 4).

XA Release in PFC Induced by Electrical Stimulation is aCalcium-Dependent Phenomenon

In these studies, a group of rats was subjected to electricalstimulation in the VTA and XA release was measured in the frontalcortex. Baseline was 19 pmol/5 min and after electrical stimulation, XArelease was 70–75 pmol per 5 minutes of dialysis. The same experimentwas repeated, but in this case the dialysis medium did not containcalcium ions, but 2.0 mM EGTA. Under these conditions, baseline XArelease was 10 pmol/5 min and electrical stimulation did not induce anyincrease in XA release (FIG. 5).

XA release in PFC After Electrical Stimulation is Inhibited byTetrodotoxin, a Sodium-dependent Sodium Channel Blocker

This study was conducted on control rats in which XA release wasmeasured in PFC after electrical stimulation. The same experiments werethen repeated, but after placing 2.0 μM tetrodotoxin in the dialysisprobe for 10 minutes. In these latter conditions, XA release induced byelectrical stimulation was completely blocked (FIG. 6).

2. Heterogeneous Distribution of XA in Rat Brain After PeripheralAdministration

Methods: Rats were given an i.p. injection of XA 50 mg/kg and thensacrificed 30 minutes later by microwave irradiation. Brains weredissected and XA was assayed in 20 different regions of the brain.

Results: The results are presented in the following table (ng/g of braintissue).

Physiological Brain structure concentrations After injection Foldincrease Prefrontal cortex 146 ± 90 1297 ± 351 9-fold (**) Frontalcortex <50 1448 ± 165 30 (***) Parietal cortex 416 ± 23 2565 ± 692  6(*) Temporal cortex 110 ± 60  995 ± 360  9 (*) Caudate-Putamen 48 ± 4 595 ± 294 12 (*) Amygdala 297 ± 13 1204 ± 180  4 (*) Substantia nigra,VTA 397 ± 73 1307 ± 351  3 (*)

XA accumulated primarily in the frontal and prefrontal cortex (PFC),temporal and parietal cortex, amygdala, dopaminergic nuclei and in thestriatum.

Among the different brain structures studied, the following regions didnot show significant accumulation of xanthurenic acid: occipital cortex,cingulate cortex, entorhinal cortex, retroslenial cortex, olfactivebulbs, pons, hippocampus, medulla oblongata, cerebellum, thalamus,nucleus accumbens, septum, globus pallidus and hypothalamus.

3. Physiological Concentrations of XA in Different Regions of Rat Brain

Methods: Animals were sacrificed by microwave irradiation and brainswere rapidly dissected on a glass slide. Numerous brain structures andnuclei were isolated and stored in liquid nitrogen until analysis. Afterweighing each structure, the tissues were homogenized in 10 volumes ofperchloric acid (mN) and centrifuged. The supernatants were analyzed byHPLC with electrochemical detection. The limit of detection is 0.05nmoles/g wet weight.

Results: The results are given in FIG. 7. They reveal thatconcentrations are heterogeneous, being particularly high in thecerebellum and olfactory bulb.

4. Identification and Characterization of Xanthurenic Acid Binding Sitesin Rat Brain

Protocol for preparation of synaptic membranes:

Synaptic membranes were prepared according to the protocol describedbelow:

-   1) Whole brains from two male Wistar rats were removed rapidly    (decapitation) and weighed.-   2) Brains were homogenized in a volume of solution S equal to 10×    the weight:    -   S=0.32 M sucrose        -   10 mM KH₂PO₄ pH 6.0        -   1 mM EDTA-   3°) Homogenizates were centrifuged at 915 g at 4° C. for 10 minutes    (Du Pont Instruments, Sorvall RC-5B) and the supernatant recovered.-   4°) Centrifugation at 18,200 g at 4° C. for 20 minutes (Du Pont    Instruments, Sorvall RC-5B).-   5°) The pellet was removed and synaptosomes were ruptured in a    volume of distilled water at 0° C. equal to 70× the weight. Polytron    for 30 seconds at maximum speed.-   6°) Distribution into Beckman centrifugation tubes according to    volume and centrifugation at 51,000 g at 4° C. (Beckman,    ultracentrifuge L8-70M) for 20 minutes.-   7°) The pellets were washed in 50 mM KH₂PO₄ buffer pH 6.0 at 0° C.-   8°) Distribution into Beckman centrifugation tubes according to    volume and centrifugation at 51,000 g at 4° C. (Beckman,    ultracentrifuge L8-70M) for 20 minutes.-   9°) The pellets were recovered and stored at −80° C.    Part 1: Study of the Pharmacological Characteristics of the    Xanthurenic Acid Binding Site Without Ions    Binding Protocol    Standard Protocol:

Binding was carried out in 50 mM Pipes buffer pH 7.4 at 0° C. (on ice)in the presence of synaptic membranes (from 0.1 to 0.3 mg of protein pertube), tritium-labelled xanthurenic acid ([³H]-XA) at variableconcentration according to type of experiment, and either buffer (todetermine total binding: TB), or “cold”, non-radiolabelled xanthurenicacid (Sigma) at 2 mM concentration (to determine nonspecific binding:NSB). Specific binding (SB) was calculated by subtracting nonspecificbinding from total binding. The incubation time was 25 minutes. Thefiltration by which free [³H]-XA was separated from [³H]-XA bound to itsbinding site(s) was carried out by rapid aspiration of the incubationmedium through Whatman (GF/B) fiberglass filters which were thensuccessively washed twice with cold 50 mM Pipes buffer pH 7.4 (2×3 mlaltogether). Filters were placed in scintillation vials to which 5 ml ofRotiszint® (Roth) were added. Vials were counted in a liquidscintillation counter (Beckman LS6000sc).

Protocol for Determining the Effect of pH:

Binding was carried out according to the standard protocol: theincubation medium was prepared from 50 mM Pipes buffer at different pH(pH studied: 5.5; 6.0; 6.5; 7.0; 7.5; 8.0). The concentration oftritiated xanthurenic acid ([³H]-XA) was 200 nM and, for determinationof nonspecific binding, non-radiolabelled xanthurenic acid (XA) was usedat 2 mM concentration. Nonspecific binding was subtracted from totalbinding to give specific binding, which varied according to pH. Theresults shown in FIG. 8 indicate that the optimum pH for xanthurenicacid binding is 7.4–7.5.

Linearity Study Versus Protein Concentration:

Binding was carried out according to the standard protocol: theincubation medium was prepared from 50 mM Pipes buffer pH 7.4 (optimumpH). The amount of protein was varied from 0.04 to 0.5 mg per tube. Thetritiated xanthurenic acid ([³H]-XA) concentration was 200 nM and fordetermination of nonspecific binding, non-radiolabelled xanthurenic acid(XA) was used at 2 mM concentration. Nonspecific binding was subtractedfrom total binding to give specific binding, which varied according tothe amount of protein: the results are linear up to 0.5 mg of proteinper tube. The results are given in FIG. 9. Subsequent experiments werecarried out using from 0.15 to 0.3 mg of protein per tube.

Measurement of Kinetic Binding Constants:

Association Constant (k₁ or k_(on)):

Binding was carried out according to the standard protocol: theincubation medium was prepared from 50 mM Pipes buffer pH 7.4 (optimumpH). The tritiated xanthurenic acid ([³H]-XA) concentration was 200 nMand for the study of nonspecific binding, non-radiolabelled xanthurenicacid (XA) was used at 2 mM concentration. The synaptic membranepreparation was added to each tube and rapid filtration was performed atthe different time points studied; the incubation times ranged from 1minute to 40 minutes. Nonspecific binding was subtracted from totalbinding to give specific binding, which increased over time to reach anequilibrum: equilibrium was attained in about 10–15 minutes. Analysis ofthe exponential association equation using Graphpad Prism software gavean observed K_(ob) expressed in min⁻¹ which is not the same as k_(on).The value of k_(ob) is 0.53±0.16 min⁻¹ (FIG. 10).

Equation used for calculation of k_(on):

$k_{on} = {\frac{k_{ob} - k_{off}}{\lbrack{radioligand}\rbrack}\mspace{14mu}{in}\mspace{14mu} M^{- 1}\min^{- 1}}$

Dissociation Constant or (k₁ or k_(off)):

Binding was carried out according to the standard protocol: theincubation medium was prepared from 50 mM Pipes buffer pH 7.4 (optimumpH). The synaptic membrane preparation was incubated withtritium-labelled xanthurenic acid (200 nM) for 25 minutes (totalbinding) after which non-radiolabelled xanthurenic acid (2 mM) was addedand incubated for different times to observe dissocation, followed byrapid filtration; incubation times ranged from 1 minute to 45 minutes.Initially, [³H]-XA is bound to its binding site, equilibrium is reached,then the longer the incubation time with non-radiolabelled XA, thegreater the decrease in binding, which reflects the dissociation of the[³H]-XA-binding site complex. Dissociation is rapid and analysis of theexponential dissociation equation using Graphpad Prism software gave adissociation constant k_(off) expressed in min⁻¹. The value of k_(off)is 0.33±0.07 min⁻ (FIG. 11).

These two constants allow an approximate extrapolation of the value forK_(d)=k_(off)/k_(on)=330 nM.

Saturation Experiment, Actual Measurement of K_(d) and B_(max):

Binding was carried out in 50 mM Pipes buffer pH 7.4 at 0° C. (on ice)in the presence of synaptic membranes (from 0.1 to 0.3 mg of protein pertube), tritiated xanthurenic acid and either buffer (to determine totalbinding), or 2 mM non-radiolabelled xanthurenic acid (to determinenonspecific binding). The incubation time was 25 minutes, followed byfiltration.

The concentration of tritium-labelled xanthurenic acid was graduallyincreased so as to reach maximal occupation of the binding sites(saturation plateau). The same thing was done in the presence of anexcess of non-radiolabelled xanthurenic acid so as to determinenonspecific binding of [³H]-XA. Then, nonspecific binding was subtractedfrom total binding to give the specific binding of xanthurenic acid toits binding site(s). Analysis with Graphpad Prism software gave theaffinity constant of xanthurenic acid for its binding site(s): Kd=743nM±250 nM. Similarly, Graphpad Prism allowed determination of the numberof binding sites present in a synaptic membrane preparation obtainedfrom whole rat brain: B_(max)=6.9±1.2 pmol/mg of protein (FIG. 12).

Competition Experiment, Determination of IC₅₀ (50% InhibitoryConcentration):

Binding was carried out in 50 mM Pipes buffer pH 7.4 at 0° C. (on ice)in the presence of synaptic membranes (from 0.1 to 0.3 mg of protein pertube), tritiated xanthurenic acid (200 nM) and either buffer (todetermine total binding), or 2 mM non-radiolabelled xanthurenic acid (todetermine nonspecific binding), or different concentrations of anon-radiolabelled molecule. If the molecule reversibly binds to the[³H]-XA binding site, the two ligands will compete with each other and adisplacement curve of [³H]-can be plotted against the concentration ofthe competitor molecule. The incubation time was 20–25 minutes afterwhich filtration was carried out.

The radioligand [³H]-XA was prepared by catalytic hydrogenation of5,7-dichloro-8-hydroxyquinoline-2-carboxylic acid in the presence ofpalladium/charcoal (10%) in methanol and in the presence of tritium gas.

After purification by HPLC, the compound had an activity of 10 Ci/mmole.

Graphpad Prism software was used to compute the IC₅₀ which is theconcentration of the compound that produces 50% inhibition ofxanthurenic acid binding. In a competition experiment between [³H]-XAand non-radiolabelled XA, a binding displacement curve was obtainedwhich, after analysis by Graphpad Prism software, gave a two sitebinding model, one IC₅₀ at 300 nM and one IC₅₀ at 57 μM. This protocolmay be used to perform competition experiments with synthetic compoundsand thereby to identify or characterize agonists or antagonists, inparticular displaying higher affinity for the xanthurenic acid receptorthan XA itself.

To this end, before conducting a classical competition experiment todetermine an IC₅₀, a screening test is first performed, that is to say,the compounds are tested at a relatively low concentration (10 μM) fortheir ability to displace 200 nM [³H]-XA (using non-radiolabelled XA ascontrol) and one looks to see whether this concentration causes the sameor more displacement than this same concentration of non-radiolabelledXA. If so, then an IC₅₀ is determined.

The [³H]-XA binding displacement study of tryptophan metabolites(L-kynurenin, 3OH-D,L-kynurenin, 5-hydroxy-L-tryptophan, picolinic acid,3-hydroxyanthranilic acid) which include xanthurenic acid was alsocarried out by screening at 200 μM concentration. None of thesecompounds caused significant displacement of xanthurenic acid. On theother hand, the screening test on kynurenic acid showed no displacementof [³H]-XA from its binding site(s) but rather a potentiation of thisbinding. In fact, binding of tritum-labelled xanthurenic acid was higherthan what was observed under normal binding conditions.

Effect of Ions on Xanthurenic Acid Binding to its Binding Site(s):

The study of the effect of ions on xanthurenic acid binding to itsbinding site(s) was carried out in 50 mM Tris maleate buffer pH 6.5 at0° C. (on ice) in the presence of synaptic membranes (from 0.1 to 0.3 mgof protein per tube), tritiated xanthurenic acid (200 nM) and eitherbuffer (to determine total binding), or 2 mM non-radiolabelledxanthurenic acid (to determine nonspecific binding) and ions at 1 mMconcentration. Incubation time was 25 minutes. The following ions weretested: Cu²⁺ (CuCl₂); Zn²⁺ (ZnCl₂); Mg²⁺ (MgCl₂); Mn²⁺ (MnCl₂); Cd²⁺(CdCl₂); Sn2+ (SnCl2), Fe³⁺ (FeCl₃).

The EC₅₀ curves for the different ions (FIG. 14) give preference tocopper ions, which have an EC₅₀ of approximately 100 μM whereas zincions have an EC₅₀ of about 500 μM. A copper ion concentration of 100–200μM roughly corresponds to endogenous concentrations in brain.

Moreover, during our research on endogenous ligands in brain that mightplay a role at the XA binding site or at a modulating site of thisreceptor, we observed that adenine and its derivatives [adenosine,adenosine diphosphate (AD P) or adenosine triphosphate (ATP)] showedsignificant affinity for the xanthurenic acid site and inhibited thebinding of tritiated XA. It is highly likely that such derivatives playan important role in modulating the activity of the XA site in brain.Such molecules may represent models for preparing structurally analogousligands that can interfere with XA sites for the development oftherapeutic or pharmacological tools.

Part 2: Study of the Pharmacological Characteristics of the XanthurenicAcid Binding Site in the Presence Of Copper Ions (Cu²⁺)

Standard Binding Protocol (Protocol Established from the pH, Linearity,Kinetic Constant and Saturation Studies):

Binding was carried out in 50 mM Tris maleate buffer pH 6.5 at roomtemperature in the presence of synaptic membranes (from 0.1 to 0.25 mgof protein per tube), tritiated xanthurenic acid ([³H]-XA) at variableconcentration according to the type of experiment, 200 μM CuCl₂ andeither buffer (to determine total binding: TB) or “cold”,non-radiolabelled xanthurenic acid (Sigma) at 2 mM concentration (todetermine nonspecific binding: NSB). Nonspecific binding was subtractedfrom total binding to give specific binding: SB. The incubation time was25 min. The filtration by which free [³H]-XA was separated from [³H]-XAbound to its binding site(s) was carried out by rapid aspiration of theincubation medium through Whatman (GF/B) fiberglass filters which werethen successively washed twice with 50 mM Tris maleate buffer pH 6.5 atroom temperature (2×3 ml altogether). Filters were placed inscintillation vials to which 5 ml of Rotiszint® (Roth) were added. Vialswere counted in a liquid scintillation counter (Beckman LS6000sc).

Effect of pH:

Binding was carried out according to the standard protocol: theincubation medium was prepared in 50 mM Tris maleate buffer at roomtemperature at different pH: 5.5; 6.0; 6.5; 7.0; 7.5; 8.0). Theconcentration of tritiated xanthurenic acid ([³H]-XA) was 200 nM (+20 μMxanthurenic acid) and, for the study of nonspecific binding,non-radiolabelled xanthurenic acid (XA) was used at 2 mM concentration.The incubation medium contained 200 μM CuCl₂. Nonspecific binding wassubtracted from total binding to give specific binding, which variedaccording to pH (FIG. 15): the optimum pH for xanthurenic acid bindingis 6.4–6.5.

Linearity Study Versus Protein Concentration:

Binding was carried out according to the standard protocol: theincubation medium was prepared from 50 mM Tris maleate buffer pH 6.5(optimum pH) at room temperature. The amount of protein was varied from0.07 to 0.4 mg per tube. The tritiated xanthurenic acid ([³H]-XA)concentration was 200 nM and, for the study of nonspecific binding, 2 mMnon-radiolabelled xanthurenic acid (XA) was used. The incubation mediumcontained 200 μM CuCl₂. Nonspecific binding was subtracted from totalbinding to give specific binding, which varied according to proteinconcentration: the results are linear up to 0.25 mg of protein per tube(FIG. 16). Subsequent experiments were carried out using between 0.10and 0.25 mg of protein per tube.

Measurement of the Copper Dose/Effect on Xanthurenic Acid Binding:

Binding was carried out according to the standard protocol: theincubation medium was prepared from 50 mM Tris maleate buffer pH 6.5(optimum pH) at room temperature. The copper (CuCl²) concentration wasvaried from 3.10⁻⁷ M to 3.10⁻⁴ M. The tritiated xanthurenic acid([³H]-XA) concentration was 400 nM (+20 μM xanthurenic acid) and, forthe study of nonspecific binding, 2 mM non-radiolabelled xanthurenicacid (XA) was used. Analysis of the dose-effect curve by Graphpad Prismsoftware gave an EC₅₀ (50% effective concentration) of 33.4 μM with aHill coefficient=1.8 (FIG. 17).

Saturation Experiment, Actual Measurement of K_(d) and B_(max)

Binding was carried out in 50 mM Tris maleate buffer pH 6.5 at roomtemperature in the presence of synaptic membranes (from 0.10 to 0.25 mgof protein per tube), tritiated xanthurenic acid and either buffer (todetermine total binding), or 2 mM non-radiolabelled xanthurenic acid (todetermine nonspecific binding). The incubation time was 25 minutes,followed by filtration.

The concentration of tritium-labelled xanthurenic acid was graduallyincreased so as to reach maximal occupation of the binding sites(saturation plateau). The same thing was done in the presence of anexcess of non-radiolabelled xanthurenic acid so as to determinenonspecific binding of [³H]-XA. Then, nonspecific binding was subtractedfrom total binding to give the specific binding of xanthurenic acid toits binding site(s). Analysis with Graphpad Prism software gave theaffinity constant of xanthurenic acid for its binding site(s):K_(d)=7.56 μM±0.8 μM. Similarly, Graphpad Prism allowed determination ofthe number of binding sites present in a synaptic membrane preparationobtained from whole rat brain: B_(max)=581.8±33 pmol/mg of protein (FIG.18).

Competition Experiment, Determination of IC₅₀ (50% InhibitoryConcentration):

Binding was carried out in 50 mM Tris maleate buffer pH 6.5 at roomtemperature in the presence of synaptic membranes (from 0.1 to 0.3 mg ofprotein per tube), tritiated xanthurenic acid (200 nM) and either buffer(to determine total binding), or variable concentrations ofnon-radiolabelled xanthurenic acid (10⁻³ M to 10⁻¹⁰ M). Graphpad Prismsoftware was used to compute the IC₅₀ which is the concentration of thecompound that produces 50% inhibition of xanthurenic acid binding. In acompetition experiment between [3H]-XA and non-radiolabelled XA, abinding displacement curve was obtained which, after analysis byGraphpad Prism software, gave a two site binding model, one IC₅₀=1 μMand one IC₅₀=114 μM (FIG. 19).

Study of the Regional Distribution of XA Binding Sites in Rat Brain byQuantitative Autoradiography Followed by Image Analysis

Method: Brains from three adult male Wistar rats were rapidly dissectedafter decapitation and frozen in isopentane kept at −40° C. on dry ice.The brains were then cut with a cryostat into slices 20 μm thick. Theslices were spread on glass slides which were then rapidly dried in coldair. The slides, mounted in a frame, were then incubated for 10 minutesin 50 mM Pipes buffer pH 7.4 kept at 0° C. on ice. The slides were thenimmersed for 20 minutes in the same buffer supplemented with 200 nMradiolabelled XA. After three brief 10-second washes with Pipes bufferwithout the radiolabelled ligand, the slices were dried in a stream ofcold air. They were then exposed to tritium-sensitive film in the dark.After two months' exposure in airtight cassettes, the films weredeveloped and shades of gray were digitized and compared with anarbitrary tritium radioactivity scale, calibrated in Curies per gramequivalent tissue (Amersham). The results, which represent thedistribution of XA receptor density in various regions of rat brain,within a factor of one, are given in the following table.

Regional distribution of [³H] xanthurenic acid binding sites. (Serial 20μm slices of brain, Wistar rats). Integrated densitometric value Brainstructure nCi/g ± SD (n = 3) Lateral caudate nucleus 195 ± 3.1 1942.6 ±30.3 252.0 ± 4.0 fmol/mg Compact substantia nigra 126 ± 2.0  806.5 ±13.8 104.6 ± 1.8 (A₉) Interpeduncular nucleus 127 ± 4.3  822.9 ± 17.3106.8 ± 2.2 Central amygdala nucleus 194 ± 4.3 1921.3 ± 42.9 249.4 ± 5.6Dorsal hippocampus 203 ± 9.8   2068 ± 41.1 268.5 ± 5.3 Ventralhippocampus 128 ± 2.1  847.5 ± 16.9 110.0 ± 2.2 Mediodorsal thalamic 172± 3.5 1568.8 ± 39.3 203.7 ± 5.1 nucleus Median post. thalamic 180 ± 7.5  1701 ± 69.9 220.9 ± 9.0 nucleus Dorsomedian hypothalamus 180 ± 6.3  1700 ± 73.2 220.7 ± 9.5 Ventral tegmental 119 ± 7.5  691.8 ± 43.6 89.8 ± 5.6 area (A₁₀) Lateral nucleus accumnbens 193 ± 10 1904.9 ± 99.0247.3 ± 12.8 Dorsal raphe nucleus (B₇) 121 ± 10  724.5 ± 59.8  94.1 ±7.7 Median raphe nucleus (B₈) 110 ± 2.0  544.2 ± 16.9  70.6 ± 2.2Cerebellar lobes 180 ± 3.5 1740.9 ± 58.5 226.0 ± 7.6 Parietal cortex 184± 1.4 1757.3 ± 23.3 228.2 ± 3.0 Temporal cortex 115 ± 7.7  634.4 ± 52.3 82.4 ± 6.8 Ant. cingulate cortex 180 ± 5.2 1691.8 ± 49.5 219.7 ± 6.4Prefrontal cortex 180 ± 11.5 1691.8 ± 108.2 219.7 ± 14.0 Periaqueductalgray 125 ± 5.65  724.5 ± 40.0  94.1 ± 5.2 substance Pyriform cortex 186± 7.7 1798.3 ± 91.7 233.5 ± 11.9 Olfactive bulbs 181 ± 7.0   1703 ± 81.5221.2 ± 10.6 Medulla oblongata 149 ± 4.9 1249.1 ± 50.7 162.2 ± 6.6Globus pallidus 163 ± 7.7 1413.2 ± 67.2 183.5 ± 8.7 Lateral septumnucleus 175 ± 6.1   1609 ± 55.7 208.9 ± 7.2 Median septum nucleus 147 ±14.9   1150 ± 116.3 149.4 ± 15.1 Occipital cortex 152 ± 51   1232 ±291.7 160.0 ± 37.8

5. Study of XA Modulation of Dopaminergic Activity in the Striatonigraland Meso-Cortico-Limbic Tracts Materials and Methods

-   Animals: Adult male Wistar rats weighing 250 to 275 g were housed in    pairs in plastic cages with a standard 7 a.m.–7 p.m. light cycle and    free access to food and water. Animals studies were conducted in    accordance with the requirements of the EEC directive of 24 Nov.    1986 (86/609/EEC).-   Chemicals: XA, 6-OHDA hydrobromide, 2,4,5-trihydroxyphenylethylamine    and desipramine were purchased from Sigma. NCS-486 was produced by    the Laboratoire de Pharmacochimie of the CNRS in Strasbourg.-   Surgical protocol: An L-shaped cannula with 4 mm tip (CMA 12,    Carnegie, Sweden) was used for the experiments. The dialysis    membrane, 500 μm in diameter, was made of polycarbonate-polyether    with a 20 Kda cutoff. The probe guide was implanted in the PFC under    stereotactic guidance. Animals were anesthetized with ketamine    hydrochloride (150 mg/kg i.p.)

Lesions Induced by 6-hydroxydopamine in Dopaminergic Nuclei A9 and A10

Animals were anesthetized with ketamine (100 mg/kg i.p.). 6-OHDA wasdissolved at a concentration of 4 μg/μl in physiological serumcontaining 0.01% ascorbic acid. One microliter was injected over 2minutes into A9/A10 using a Hamilton syringe under stereotacticguidance. After the injections, the toxin was allowed to diffuse for 1minute. Noradrenergic neurons were protected by prior injection ofdesipramine 25 mg/kg i.p., 45 minutes before injection of 6-OHDA.

Four weeks later, rats received apomorphine 1 mg/kg by subcutaneousinjection and the number of controlateral rotations executed 15 minutesafter the injection was recorded.

The rats with induced lesions performed 130±45 turns/15 minutes. Sixweeks after local injection of 6-OHDA in the right A9/A10 dopaminergicnuclei, rats displaying good functional alteration were selected andimplanted for the microdialysis studies.

Microdialysis Protocol

The experiments were carried out in conscious rats with and withoutinduced lesions, 24 to 48 hours after surgical insertion of themicrodialysis probe. The composition of the microdialysis medium was asfollows: 147 mM NaCl; 1.2 mM CaCl₂; 1.2 mM MgCl₂; 4.0 mM KCl, pH 6.5.The dialysis rate was 1 μl/min (CMA 100 pump, Carnegie). Dialysates werecollected in 20 minute fractions and stored immediately in liquidnitrogen pending analysis.

Analysis of Dopamine, DOPAC and HVA in the Microdialysis Medium

These compounds were analyzed by HPLC with electrochemical detection.The chromatographic system consisted of a 25 cm×4.6 mm C18 columnmaintained at a constant temperature of 30° C. The mobile phase was 50mM NaH₂PO₄ containing 0.1 mM EDTA and 6% methanol, the pH of thesolution was adjusted to 4.85.

In Vitro Yields

The in vitro dialysis yield for dopamine was previously determined to be16% at laboratory temperature and for a dialysis rate of 1 μl/minute.

Histology

After the experiments, the correct positioning of the probe was alwayschecked by post-mortem histological examination of the brain afterfixation in paraformaldehyde.

Statistical Analyses

The microdialysis experiments were statistically analyzed by an ANOVAfollowed by a Newman-Keuls test of multiple comparisons.

Results

Dose/effect of Local XA Infusion in the PFC on Extracellular DopamineConcentrations

Retrodialyzed XA concentrations were respectively 1 μM (black circles),5 μM (black squares) and 20 μM (black triangles). XA was applied duringa 20 minute dialysis. The results are expressed as the percentage ofmean baseline dopamine release (determined on eight 20-min dialysisfractions prior to stimulation by XA) (FIG. 20).

The lowest XA concentration (1 μM) had no effect on dopamine release. Onthe other hand, the 5 μM concentration (approximately 1.5 μM XA in braintissue based on in vitro yields) increased extracellular dopaminerelease by approximately 250%. The 20 μM concentration (about 6 μM intissue) increased dopamine release by 400–450%.

Effects of Local Application of 20 μM XA in the PFC on Release ofDopamine, DOPAC and HVA

The results are expressed as the percentage of dopamine release relativeto baseline (determined on eight 20-min dialysis fractions prior toinjection of XA), FIG. 21. Black squares represent dopamine, blackcircles represent DOPAC and white squares represent HVA. In this case,as seen previously, dopamine release was increased by 400–450% frombaseline after 20 minutes, but DOPAC and HVA release decreased slightlyafter 100 to 120 minutes.

Effects of Local Application of 20 μM XA in the PFC on ExtracellularDopamine Release, in the Presence or Absence of 20 μM NCS-486

The results are expressed as the percentage of dopamine release relativeto baseline (mean of eight 20-min dialysis fractions prior to injectionof XA) (FIG. 22: application of 20 μM XA for 20 minutes (black circles),or co-infusion of 20 μM XA+20 μM NCS 486 (white squares) or applicationof 20 μM NCS-486 alone (white triangles)). It can be seen that treatmentwith NCS-486 alone or with NCS-486+XA did not modify dopamine release,while XA alone increased dopamine release by approximately 300%, about40 minutes after the stimulation.

6. Electrophysiological Studies

Electrophysiological Recordings

The neuron cell line NCB-20 was studied by using the patch-clamptechnique (Hamill et al., 1981) in the so-called cell-attached patchconfiguration. The recording media were designed so that only theactivity of single aspecific cation channels and chloride channelslocated in the membrane fragment under the pipette would be recorded.Specific calcium, sodium and potassium currents were minimized byreplacing the majority of cations in the pipette medium by an impermeantcation, N-methyl-D-glucamine (NMDG) or blockage by TEA(tetra-ethyl-ammonium). Replacement of permeant cations in the pipetteby NMDG also made it possible to have aspecific cation currents mainlyin the outflux direction since the influx component was minimized.Furthermore, in some experiments, chloride channel activity was recordedonly in the influx direction by replacing chloride ions in the pipettemedium by TCA (trichloroacetic acid). Control of cell membrane potentialat a value close to zero mV was achieved by using an extracellular KClmedium. Thus the potential of the recorded membrane fragment correspondsto the opposite of the potential imposed in the recording pipette. Underthese conditions the recording media had the following composition (inmM): NMDG/Cl (or TCA) 140, KCl 2, MgCl₂ 1, HEPES 10, TEA/Cl 15, for thepipette medium and NaCl, KCl 141, CaCl₂ 0.5, HEPES 10, EGTA 5, for thebath; in both cases the pH was adjusted to 7.4 with TRIS base and KOH,respectively.

Chemicals were from Sigma (Saint Quentin Fallavier, France).

After making a gigaseal with pipettes having a resistance comprisedbetween 4 and 7 MΩ, ion currents were measured with a patch-clampamplifier (EPC-7 amplifier, List-Medical, Darmstadt, Germany or AxopatchB200, Axon Instruments, CA). Signal acquisition (acquisition frequencyfrom 1 to 5 kHz) was then done using an interface card (ScientificInstruments, OH) and pClamp 6 software (Axon Instruments, CA). Theproducts were diluted to the desired concentration in the extracellularmedium. Application was by gravity through a multipore perfusion systemwith an internal pore diameter of 300 μm. The solution was changed byplacing the corresponding pore opposite the recorded cell after openingthe valve.

Results

NCB-20 cells express a binding site specifically recognized byxanthurenic acid. These functional experiments aimed to detect anyelectrogenic membrane phenomenon that might be activated by xanthurenicacid receptor(s). There is some evidence suggesting that this receptoris coupled to G proteins and that a membrane effect would thereforeoccur through an intracellular relay. Accordingly, we investigated aneventual action on cation and chloride channels, whose activationdepends on both membrane potential and cystolic factors (mainly thecalcium ion and kinase proteins; Evans and Marty, 1987; Taleb et al.,1988; Leech et al., 1996). These two types of ion channel were thereforerecorded singly (see methods) and by imposing one or the other of thetwo permeabilities. The inversion potential of the response would becomprised between −49.5 or −11.8 mV, respectively, for a cationic(monovalent cations) or chloride response. Under these conditions anychange in cytoplasmic factors will also be revealed by the parameterscharacterizing the current-potential relationship (FIG. 23).

Cells were recorded in the cell-attached patch configuration. Thecurrent crossing the channels inserted in the membrane under the pipettewas recorded singly. The cell membrane potential was set at 0 mV by aKCl concentration in the extracellular medium equivalent to that of thecytoplasm. Under these conditions the potential of the membrane fragmentunder the pipette is opposite to that of the pipette.

Effect of Xanthurenic Acid on Activation of the Cation Current

The membrane fragment under study was periodically stimulated (frequency0.2 Hz) by a potential gradient of −70 to 100 mV (see protocol in FIG.24A, lower trace). In control conditions using a pipette medium depletedof chloride ion (replaced by TCA), the recorded current was stable andhad an amplitude of −0.4±0.1 and 8.6±0.2 μA (n=30) at potentials of −70and 100 mV, respectively. This current reversed at a potential of48.3±3.5 mV (n=3). This value, which is close to the inversion potentialof monovalent cations (−49.5 mV, if intracellular concentrations of K⁺and Na⁺ are estimated at 140 and 2 mM, respectively), shows that underthese recording conditions, there is a selective basal permeability tocations. The current-potential relation shows a marked outfluxrectification (FIGS. 24A and C), in agreement with the asymmetricaldistribution of cations on either side of the membrane fragmentrecorded. In the presence of xanthurenic acid at concentrations of 1 to20 μM after a latency (from 1 to 4 minutes) dependent on the agonistconcentration, the amplitude of the cation current increased by 10 to40% with a slow kinetics to which can be added transient large amplitudecurrents. In the case of FIG. 24, the current amplitude at the peak ofthe transient current and at 100 mV potential was increased by a factorof 5.

On average, for an agonist concentration of 20 μM, the current amplitudeincreased by a factor of 5.0±1.7 (n=3). The I-V relationship of thecurrent specifically activated in the presence of xanthurenic acid(after subtracting the current observed in control conditions) wassimilar to that of the control current (FIG. 24C). In particular, theinversion potential was virtually identical to that of the control andhad a mean value of −51.6±2.1 (n=3). This is true for both the slowcurrent and the transient current. This inversion potential is close tothe equilibrium potential of monovalent cations suggesting that in theseconditions, xanthurenic acid can induce the activation of a current ofthe cationic type. The observation that such current is recorded on amembrane fragment physically isolated from the rest of the cell wherethe agonist is applied, indicates that activation of such cationchannels occurs through an intermediary intracellular relay between thereceptor and the ion channel.

To answer the question of whether such cation channels also allowpassage of divalent cations, calcium in particular, we performed thesame type of experiment as hereinabove but with a pipette mediumcontaining mainly the Ba²⁺ ion (all monovalent cations were replaced byBa²⁺ except 2 mM K⁺ ions and 15 mM TEA⁺ ions). Under these conditions aswell, in the presence of xanthurenic acid we observed the activation ofthe current whose inversion potential in this case had a mean value of2.6±2.5 mV (n=7). Replacing monovalent cations by Ba²⁺ shifted theinversion potential of the response by approximately 54 mV. In the casewhere Ba²⁺ ions would not participate in the current activated by theagonist, the predicted inversion potential would have a value of −53.5mV. The observed difference reflects the passage of Ba²⁺ ions throughthe cation channels.

Effect of Xanthurenic Acid on Activation of a Chloride Current

On some responses, however, particularly those not showing a transientcurrent, we observed a variation in the inversion potential of thecurrent induced by the agonist. For instance, in the case illustrated inFIG. 25, the onset of the response had an inversion potential of −16.5mV which reflects contamination of the cation current by another currentwhose equilibrium potential has a more depolarized value. In a secondphase of the response the potential has shifted towards an even moredepolarized value of −4.2 mV. The added current has an inversionpotential compatible with a movement of chloride ions in ourexperimental conditions.

Pharmacology of the Response to Xanthurenic Acid

NCS-482 applied at a concentration of 10 to 20 μM to NCB-20 cellsinduced a response virtually identical to that of xanthurenic acid(FIGS. 25 and 26). This result indicates that NCS-482, a xanthurenicacid derivative able to displace xanthurenic acid binding, acts as anagonist of the xanthurenate receptor. NCS-486, another xanthurenic acidderivative which also displaces xanthurenic acid binding, had no effectwhen applied alone (20 μM), whereas it reduced the amplitude of theagonist-induced response (FIG. 26), which makes it a xanthurenatereceptor antagonist.

7. Neuropharmacology of Xanthurenic Acid (XA): Behavioral Studies in theRat

1) Infrared Cell Measurements of Locomotor Activity

The following doses were studied: 12.5, 25, 37.5, 50, 100 and 200 mg/kg(i.p.) (FIG. 27). This test demonstrated a decrease in motor activity,and this at XA doses greater than or equal to 50 mg/kg.

The intensity of sedation was proportional to the injected dose.

2) Measurement of Global Reactivity of the Animals by the “Open Field”Test

This test confirmed the absence of sedation at a dose of 37.5 mg/kg, thedose at which exploration of the center of the cage was significantlyincreased, which might be interpreted as a potential anxiolytic effect(FIG. 28). This tendency was confirmed by a test evaluating socialinteractions between two congeners (Ramos et al., Behav. Brain Res.,1997, 85: 57–69). The 50 mg/kg dose produced a significant increase inthe duration of social interactions in treated animals as compared tountreated control congeners. At the 100 mg/kg dose, this evaluation washindered by a significant sedative effect induced by the product.

3) Demonstration of an Effect of the Antidepressant Type by the PorsoltTest

The following doses were used: 50, 100, 150 and 200 mg/kg i.p.

This test has been validated with imipramine 30 mg/kg. The testdescribed by Porsolt et al., Eur. J. Pharmacol. 1978, 47: 379–391, wasused.

Doses of 100, 150, 200 mg/kg produced a statistically significantdecrease in the cumulative duration of immobility, indicating that theeffect obtained was similar to that of imipramine (FIGS. 29 and 30).

4) Demonstration of an Effect of Xanthurenic Acid on MemorizationProcesses by the Object Recognition Test

Ennaceur and Delacour, Behav. Brain Res. 1988, 31: 47–59.

Doses of 37.5 and 75 mg/kg i.p. were used.

The results (FIG. 31) revealed a significant increase in the objectrecognition score for treated animals as compared to controls. Theincrease in the score can be interpreted as a beneficial effect of theproduct on short-term memory.

5) Effects of Xanthurenic Acid After Intracerebral Injections

The product was injected by the intracerebroventricular route (i.c.v.)on the one hand, and in defined brain structures on the other hand:ventral tegmental area (A10), substantia nigra (A9) or nucleusaccumbens. At XA doses of 2, 4, 10, 50 μg/rat, dose-related stereotypieswere observed. After disappearance of the stereotypies (30±15 minutes),reinjection of XA induced the same effects.

6) Study of the Antagonist Potential of NCS-486 on Animals WithUnilateral Lesions of the Striatonigral and Mesocorticolimbic TractsInduced by Intracerebral Injection (VTA; SNc) of a Neurotoxin (6-OHDA)After Protection of Noradrenergic Neurons by Pretreatment WithDesipramine

Injection of a non-sedative dose of XA (25 mg/kg i.p.) did notantagonize the controlateral rotations induced by apomorphine (0.05, 0.1mg/kg s.c.) Studies of the potentiation of ipsilateral rotations inducedby amphetamine and possible antagonism by NCS486 of ipsilateralrotations induced by xanthurenic acid 25 mg/kg i.p. are under way.

7) Electroencephalographic (EEG) Analysis of Sedation Induced by i.p.Injection of Xanthurenic Acid

Doses tested ranged from 50 to 500 mg/kg i.p. The detailed EEG analysisdid not show evidence of “epileptogenic” or “deep NREM sleep” activity.Only a reduction in the amplitude of the EEG waves was observed. Thissedation was accompanied by a significant, dose-related decrease in bodytemperature (−0.5 to −2° C.).

IN CONCLUSION, the neuropharmacological studies conducted in ratstreated with xanthurenic acid either by peripheral or localadministration, showed the following:

XA has sedative activity in animals, with a very clearcut dose-effectrelationship. Such sedative effect is corroborated by several tests. Inthe open field test which evaluates the spontaneous behavior of theanimals, XA was found to exert an anxiolytic effect at non-sedativedoses and it promoted social interactions. Certain tests aiming toevaluate an antidepressant effect of XA revealed a dose-dependent moodenhancement in animals. Memory tests showed that XA promotes short-termmemory. Finally, this substance has potent dopaminergic activity asdemonstrated by the induction of stereotypies. Such stereotypies areblocked by high doses of the antagonist NCS-486. EEG studies showed thatXA has no epileptogenic effects and does not induce deep NREM sleep,although a decrease in the amplitude of the EEG waves and hypothermiawere observed, two phenomena which may be correlated with sedation.

8. Demonstration and Characterization of an XA Transport System inCultured Neurons

1) Materials and Methods

NCB-20 cell cultures

Maintenance of the cell line:

NCB-20 cells were stored frozen in liquid nitrogen as aliquots of theparent strain. For the purposes of an experiment, an aliquot was thawedand placed in culture conditions appropriate for the study. To maintainthe cell line, as soon as the culture dish reached confluence, theinitial dish was used to inoculate another dish by taking only analiquot of the cell suspension in the original dish. In this manner,subcultures were carried out in DMEM medium supplemented with 10% fetalcalf serum (FCS) and dilution was generally by a factor of ten.

Culture of Differentiated NCB-20 Cells:

The cell culture was prepared by using a culture dish of NCB-20 cells at80% confluence. The culture medium was removed and replaced with 10 mlof DMEM supplemented with 10% FCS. Cells were then detached andsuspended by repeated passage of the medium through a 2 mm gauge needle.Cells were then counted in a hemocytometer. The final suspension wasadjusted to a cell density of 3.10⁴ cells/ml in DMEM medium supplementedwith 10% FCS and 1 mM cAMP. Petri dishes were seeded with 2 ml of thissuspension and incubated at 37° C. in a moisture-saturated CO₂atmosphere. Cells were used four days after inoculation.

Measurement of XA Transport in NCB-20 Cells

The culture medium of 4-day NCB-20 cell cultures differentiated in cAMPwas replaced by one of the buffer solutions described hereinabove (Krebswith or without Na⁺, to respectively evaluate Na⁺-dependent activetransport and passive transport, which occurs mainly through diffusion)according to the type of experiment planned. Culture dishes were placedin a water bath at 37° C. for 10 minutes. The buffer was then replacedby the same buffer solution containing [³H]-XA and XA at concentrationsranging from 1 to 500 μM. [³H]-XA was present at very low concentrationand served only as a tracer of the cold (non-radiolabelled) XA. After 1minute of incubation, the solutions were aspirated and the cells washedthree times for 10 seconds with 1 ml of the same buffer (Krebs with orwithout Na⁺), kept on ice. Active transport requires energy (ATP) and ismarkedly slowed at temperatures below 37° C. The washed cell layer wasfrozen in dishes then, the next day, distilled water was added to obtaina solution of cellular debris containing the intracellular radiolabel([³H]-XA). Freezing facilitated homogenization by repeated passage ofthe solution through a pipette. A 900 μl aliquot was added toscintillation fluid (Rotiszint®, 4 ml). The remainder of the solutionwas sampled to assay protein by the BCA method (Uptima). Theradioactivity measured in the scintillation counter was converted bymeans of a suitable calibration curve into picomoles of XA taken up bythe cells per mg of protein.

The same procedure was carried out for each dish containing thedifferentiated cells. However, in each experiment, one of the parameterswas modified so as to determine the kinetic and pharmacologicalcharacteristics of XA transport in NCB-20 cells.

The conditions of transport were measured as a function of:

Incubation Time:

These experiments defined the kinetics of XA transport.

For each incubation time, transport was measured on nine dishes ofNCB-20 cells with Na⁺ and without Na⁺. Ten different time points werestudied: 0-10-30 seconds and 1, 2, 3, 5, 8, 10, 12 minutes. Fourdifferent experiments were carried out.

[³H]-XA Concentration:

These studies defined the kinetic constants: maximal velocity oftransport (V_(m)) and XA concentration required to attain half-maximalvelocity (K_(m)).

For each XA concentration, XA transport was measured on nine dishes ofNCB-20 cells with Na⁺ and nine dishes without Na⁺. Seven differentconcentrations were tested: 1-5-12.5-25-50-100-200 μM. Four independentexperiments were carried out.

Protein Assay

Protein was assayed by the microplate technique. The microplate musthave the required number of wells to perform a duplicate assay for eachculture dish and for a standard curve.

The cells at the bottom of each treated culture dish were taken up in 1ml of doubly-distilled water. Several aspirations-releases wereperformed to detach all the cellular debris and homogenize thesuspension. Two 20 μl test samples were then removed and placed in thewells of the microplate.

A space was reserved for the calibration curve ranging from 0.032 to 2mg/ml. Each suspension of cellular debris was measured in duplicate. Twohundred microliters of reagent (mixture of 50 parts reagent A and onepart reagent B) were then added to each well. The plate was shaken andincubated at 37° C. for 30 minutes.

Radioactivity Counting

Four milliliters of scintillator were added to the tubes containing 900μl of the cell suspension obtained. The control tube contained 900 μl ofdoubly-distilled water, and the tubes used to measure specific activitycontained 10 μl of the radioactive solutions used as reagent during theexperiment and 890 μl of doubly-distilled water. The tubes were closed,vortexed and left overnight at 4° C. before being counted in thescintillation counter. This count measures intracellular radioactivitywhich is directly proportional to the quantity of XA that entered thecell through passive or active transport. To calculate the amount ofactive transport, passive transport (Krebs without Na⁺) is simplysubtracted from total transport (Krebs with Na⁺).

2) Results Concerning XA Transport

All calculations were performed on the Excel computer program. Theresults were then converted to graphic form with Prism 3.0 softwarewhich also computed the equations of the line and statistical analysis.

The results concerning XA transport in NCB-20 cells over time are givenin FIG. 32.

In the study of transport as a function of incubation time of the cellsin the presence of XA, three similar-looking curves were obtained.Transport increased by about 20% in the presence of sodium. The threecurves reached a plateau after approximately 1 minute, thus defining theoptimal time at which to measure XA transport in NCB-20 cells. This1-minute time was used in subsequent experiments.

The results concerning XA transport in NCB-20 cells according to [³H]-XAconcentration are given in FIG. 33.

The Scatchard plot gives the mathematical constants of intracellulartransport of XA, with K_(m)=105 μM and V_(max)=1229 pmoles/mgprotein/minute.

In the study of XA transport as a function of XA concentration,saturation of transport and therefore of the transporter was observed,which can be mathematically defined by the value of the maximal velocityof transport, which in our experiments is V_(max)=1229±440 pmoles/mgprotein.min. Another mathematical constant which characterizes transportis the K_(m), which is the concentration of XA needed to attain half themaximal velocity. In our experiments, K_(m)=105±81 μM.

The optimal XA concentration for the pharmacological studies on thismolecule will be 100 μM, which corresponds to the calculated K_(m).

Interference of Other Endogenous Molecules with XA Transport

Other endogenous molecules which might be transported by the XAtransporter were tested. In addition, to demonstrate theenergy-dependent nature of this transport, we blocked cellular energyproduction with deoxyglucose which inhibits glycolysis. The results areshown in the histogram in FIG. 34 as the percentage entering the cellsrelative to transport in the presence of radiolabelled XA alone (=100%).

L-tryptophan, L-tyrosine and kynurenic acid were found to be goodinhibitors of cellular transport of XA. It may therefore be possiblethat uptake of XA by neurons makes use of the transporter of neutralamino acids. The presence of copper ions or zinc ions in the medium ledto a highly significant potentiation of XA transport (3-fold higher forcopper ions). The presence of the cellular poison 2-deoxyglucose causedan approximate 50% decrease in transport, indicating that transportrequires intact cellular energy production.

3) Conclusions of XA Transport Studies

The different experiments carried out reveal the presence of an activetransport system for XA in NCB-20 cells. Such transport is an activephenomenon which requires the presence of sodium ions in the reactionmedium. The transport is characterized by fairly rapid kinetics sincethe optimal velocity is reached after an incubation time of about 1minute. The kinetic constants of this active transport system weremeasured and the maximal velocity was found to be 1229 pmoles/mgprotein.min with K_(m)=105 μM. Some amino acids, including tryptophanand tyrosine, inhibit this transport, suggesting that the transporterinvolved may be a neuronal transporter for neutral amino acids and thatXA transport interferes with the transport of amino acids essential forcatecholamine and serotonin synthesis. The synthetic compounds, whichare XA receptor ligands, could be tested on this transporter to checktheir specificities.

9. Organization and Role of the XA System in the Regulation ofDopaminergic Activity

1) Is XA Co-Released With Dopamine at the Same Nerve Terminals UponStimulation of Dopaminergic Nuclei A₉–A₁₀?

To investigate this question, we studied the modifications ofextracellular XA and dopamine release that occur after inducing lesionsin dopaminergic nuclei with 6-hydroxydopamine (6-OHDA). This lesiondestroys in particular the dopaminergic nerve terminals in the frontalcortex. We further investigated the effects of XA on glutamate and GABArelease in an attempt to elucidate the sequence of events leading to themodification of dopamine release in the frontal cortex in rats.

Protocol:

A unilateral lesion in the mesencephalic dopaminergic tract was inducedby stereotactic injection of 6-hydroxydopamine hydrobromide (6-OHDA HBr,Sigma) into the ventral tegmental area (VTA) and the compact substantianigra (SNc). The procedure was performed under stereotactic guidance(Narishige) on Wistar rats under imalgene anesthesia (100 mg/kg i.p.).The neurotoxin 6-OHDA was dissolved in isotonic sodium chloride solution(0.9% NaCl) containing 0.01% ascorbic acid at a final concentration of 4μg free base per μl and 6 μg (1.5 μl per injection) were injected over 2minutes through a 20G stainless steel injection cannula. In agreementwith the Paxinos and Watson atlas, the stereotactic coordinates used areexpressed in millimeters relative to the bregma: AP: 2.3; ML: 0.5; DV:8.7 mm and AP: 2.3; ML: 2.0; DV: 7.5 mm, respectively for the VTA andSNc. The dorsoventral coordinate was taken from the cranial bone, theear bars being positioned 3.3 mm below the bar of the upper incisors.After injecting the entire volume at a rate of 1 μl/min (CMA 100 pump),the needle was left in place for 1 minute more so as to allow the toxinto diffuse freely around the injection site. Noradrenergic neurons wereprotected from damage by prior injection of desipramine 25 mg/kg i.p. 45minutes before injection of 6-OHDA.

Selection of Animals

Four weeks after induction of the lesions, the rats were tested aftersubcutaneous administration of a direct agonist (apomorphine 0.1 and 1.0mg/kg) and the number of controlateral rotations (side opposite to thelesion) in 15 minutes was recorded. The result for rats treated with theneurotoxin was as follows: 130±45 turns/15 minutes (n=12 animals).

Six weeks after the injections in the right compact substantia nigra andventral tegmental area, rats with a satisfactory result in theapomorphine-induced controlateral rotation test were selected andimplanted for the microdialysis experiments.

On completion of the experiments the animals were sacrificed. Thestriatum and ipsilateral and controlateral mesencephalic nuclei wereremoved and analyzed by HPLC for residual dopamine content.

The results, expressed in pmoles/g of wet tissue±SEM (n=3) are asfollows:

Damaged striatum: 261±123 versus intact striatum 9140±1413 (−98%p=0.0033)

Damaged VTA —SNc: 32±31 versus intact VTA —SNc 563±128 (−95% p=0.0157).

These results indicate that the lesion of dopaminergic neurons wassatisfactory and that there was a very marked decrease in dopaminelevels in the striatum and dopaminergic nuclei.

Measurement of Extracellular Release of Dopamine and its MetabolitesAfter Neurotoxin-induced Lesion of Mesencephalic Dopaminergic Nuclei:

Neurotoxin-treated animals were implanted with the dialysis probe andbipolar electrode, electrically stimulated in the VTA (100 and 200 μA)and dialyzed in the prefrontal/anterior cingulate cortex according tothe same protocol described for the previous experiments.

Among the measured parameters (DA, DOPAC, HVA, XA and 5-HIAA), only XAand 5-HIAA were detectable on the chromatograms.

The results show that electrical stimulation (100 and 200 μA) of the VTAafter 6-OHDA-induced lesion of dopaminergic nuclei (VTA and SNc) inducedXA release in the frontal cortex (+151±51% and +667±115% for stimulationwith 100 μamperes and 200 μamperes, respectively), accompanied byrelease of 5-HIAA without modifying residual dopamine release (DA, DOPACand HVA) after lesion induction.

These results demonstrate that XA and dopamine are not co-released inthe frontal area, although XA modulates dopamine release. XA istherefore released by nerve terminals which are probably specific andmodulates dopamine release via receptors situated at dopaminergicsynapses, either in the dopaminergic nuclei themselves (VTA and SNc), orin nerve terminals controlling glutamate and/or GABA release in thefrontal cortex. Quantitative autoradiography revealed the existence ofhigh-affinity XA binding sites in both the frontal cortex and inmesencephalic dopaminergic nuclei.

2) Modification of Frontal Extracellular Release of Glutamate (GLU) andGABA After Retrodialysis of 20 μM XA for 20 Minutes

The aim here was to investigate whether XA can modify glutamate and/orGABA release. Such modifications would then affect dopamine release.According to this hypothesis, XA receptor sites would be found onglutamate and/or GABA neurons in the frontal cortex.

a) Measurement of Amino Acid Concentrations in the Dialysates

Amino acids contained in 15 μl of dialysate were separated according totheir hydrophobicity by reverse phase high performance liquidchromatography (HPLC). The system comprised a degasser (Waters In-LineDegasser), a pump (Waters 626 Pump, Waters 600 S Controller), arefrigerated CMA/200 sampler-injector (Refrigerated Microsampler,CMA/microdialysis, Carnegie) fitted with a 51 μl loop, a Nucleosil C18column (5 μm, 25×0.4 cm), an oven (Waters), and a fluorimeter (Waters470 scanning fluorescence detector). The mobile phase was composed of abinary gradient between solution A: 0.05 M NaH₂PO₄ (Roth) 80% (pH 4.8adjusted with 10 N NaOH), methanol 20% (Chromanorm, Prolabo); andsolution B: 0.05 M NaH₂PO₄ 20% (pH 4.8 ajusted with 10 N NaOH), methanol80% and THF 5%. Glutamate and GABA were measured by fluorimetricdetection at an excitation wavelength of 345 nm and an emissionwavelength of 455 nm after derivatization. Chromatogram acquisition andquantitative data analysis were done with Millenium software (Waters).

The samples were derivatized by mixing 15 μl of dialysate with 15 μl ofthe following derivatization solution: 7.5 mg o-phthaldialdehyde (Sigma)in 4.5 ml of 0.1 M sodium tetraborate pH 9.3, 500 μl of methanol and 10μl of 3-mercapto-propionic acid (Sigma). The elution rate was 0.8 ml/minat 35° C. in a series of steps: 90% A and 10% B at 0 min; 40% A and 60%B to 15 min (linear gradient); 40% A and 60% B to 19 min (isocraticgradient); 0% A and 100% B to 19.1 min; 0% A and 100% B to 24 min(isocratic gradient); 90% A and 10% B to 24.1 min (isocratic gradient)and 90% A and 10% B to 30 min. The limit of detection for glutamate andGABA in the samples was 0.75 fmoles (0.05 μM). β-amino-isobutyric acid(Sigma) was used as internal standard.

Results:

Retrodialysis of 20 μM XA for 20 minutes in the frontal cortex led to animmediate 37%±1 decrease in GLU release during 90 minutes beforegradually returning to baseline. The results shown in FIGS. 35 and 36are expressed as %±SEM (n=2). The 100% value (1 nmole/20 μl) representsthe mean of four consecutive samples before stimulation.

10. Consequences of Stress Induced by Intermittent Electric Shock onNorepinephreine (NE) and XA Release in the Median Prefrontal Cortex(PFC)

This study was based on the hypothesis that norepinephrine (NE) in theprefrontal cortex coming from the locus caeruleus (A6) plays animportant role in the individual behavioral differences in reactivity tostress. As the A6 nucleus was labelled with tritiated XA in theautoradiography studies, the aim was to see whether frontal release ofXA could be involved in the mechanisms of electric shock-induced stressin animals.

Since spontaneous locomotor activity in a new environment is abehavioral indicator of reactivity to a stressor, we selected “responderanimals” exhibiting a more marked behavioral response in this test.

The main components of the stress response are extrahypothalamic releaseof CRH (corticotropin releasing hormone) leading to stimulation oftyrosine hydroxylase (TH) which results in increased norepinephrine inthe locus caerulerus and consequently increased NE release at nerveterminals (prefrontal cortex, amygdala and dentate gyrus).

Protocol:

1) Selection of Animals in the Open Field Test:

Number of squares crossed in a 5 minute session: 55.08±22.6 (6.53)

Mean±SD (SEM) n=12 Wistar rats weighing 350 g.

2) Stress and Neurochemical Quantification:

Stress was induced by intermittent electric shocks to the paw at a rateof 1 shock of 0.3 mA per min for 20 minutes.

Norepinephrine release in the prefrontal cortex was measured using theintracerebral microdialysis method on conscious animals coupled to ahigh performance liquid chromatography system with electrochemicaldetection.

Results:

The results are given in FIG. 37.

During the duration of the stress induced by intermittent electricshocks of 300 μA for 1 sec per minute for 20 minutes, norepinephrinerelease in the prefrontal cortex increased by +151% relative to baselinerelease and XA release increased by +250%.

These findings indicate that norepinephrine and XA are releasedconcomitantly in the frontal cortex of stressed animals. Release of XAmight be a stress adaptation reaction and administration of XA receptoragonists in stress situations may represent a novel therapeuticsolution, especially since dopamine release is very low and XA and XAreceptor agonists increase this release.

11. Neuropharmacological characterizations of XA Receptor Antagonists:study of the antagonist NCS-486

1) Study of Dopaminergic Stereotypies Induced by Intracerebral Injectionof XA and Reversal by the Antagonist NCS-486

“Stereotypies” are involuntary movements made by the animal afteradministration of direct (apomorphine) or indirect (amphetamine)dopaminergic agonists.

The following stereotypies are observed: licking, chewing,hyperactivity, exploring, standing, grooming, burrowing, stretching andsniffing.

Effects of Xanthurenic Acid:

Intracerebral injections of XA in the striatum, VTA, lateral ventriclesor nucleus accumbens induced dose-dependent stereotypies (dosesinjected: 2, 10, 50, 100 and 150 μg/rat). These behaviors developed twominutes after the intracerebral injection and lasted for 30 to 40minutes. One hour after such behaviors disappeared, they could bere-induced by another injection.

After neurotoxic lesion of the mesencephalic nuclei (A₉–A₁₀) induced by6-OHDA, injection of XA 25 mg/kg i.p. did not modify the number ofcontrolateral rotations induced by apomorphine 0.1 mg/kg s.c. Similarly,XA 25 mg/kg had no effect on the number of ipsilateral rotations inducedby administration of amphetamine.

Effects of NCS-486 on stereotypies induced by XA, apomorphine oramphetamine:

NCS-486 at a dose of 2 μg/rat i.c.v. completely antagonized thestereotypies induced by XA 2 μg i.c.v., whereas injection of NCS-486alone (2 μg i.c.v.) had no effect on the animals' behavior.

After i.p. or i.c.v. injection, NCS-486 did not antagonize stereotypiesinduced by apomorphine 0.5 mg/kg s.c. On the other hand, NCS-486, at adose of 200 mg/kg i.p. injected 30 minutes before XA 2 μg i.c.v.suppressed or strongly reduced the stereotypies.

2) Study of NCS-486 on General Activity of the Animals Measured in theOpen Field Test and Reversal of XA-Induced Sedation

This study showed that NCS-486 100 mg/kg p.o produced hyperactivity inthe animals 1 hour after administration. Furthermore, NCS-486 was ableto antagonize the sedation induced by i.p. injection of XA 100 mg/kg.

The results are presented in the following table:

Treated with NCS- Behavior Controls 486 Variations in % Gromming 1 ± 1 3 ± 1 — Jumping 1 ± 2  2 ± 2 — Standing 20 ± 14  32 ± 1 — Total squares55 ± 37 143 ± 2** +260 Outer squares 51 ± 33 128 ± 21* +251 Innersquares 4 ± 4  14 ± 1* +350 Defecations 2 ± 1  0 — The results are givenas the mean ± SD (n = 4 rats/group) Student's test: **p < 0.05; **p <0.01

Study of reversal of sedation induced by XA 100 mg/kg i.p

The results are presented in the following table:

XA 100 mg/kg XA 100 mg/kg (i.p.) + ND-7002 Behavior Controls (i.p.) 100mg/kg (per os) Grooming  4 ± 1  4 ± 2  4 ± 1 Standing  66 ± 4  37 ± 14 51 ± 11 Total squares 193 ± 22 110 ± 25* 182 ± 15 Outer squares 153 ±18  91 ± 17* 148 ± 15 Inner squares  39 ± 9  19 ± 6  34 ± 8 The resultsare given as the mean ± SEM (n = 4 rats/group) Student's test: *p < 0.05

12. Characterization of the Neuropharmacological Activity of LigandsBinding to the Allosteric Site for Kynurenic Acid, an Integral Part ofthe XA Receptor Site: Example of Compound ND-7000, Derivative ofXanthurenic Acid or Kynurenic Acid

Effects of ND-7000 on stereotypies induced by XA.

ND-7000 administered by intracerebroventricular injection at a dose of20 μg/rat did not induce any stereotypies in the animals. At a dose of50 μg/rat, ND-7000 induced considerable sedation. In contrast,simultaneous injection of 10 μg of ND-7000 and 10 μg of XA led to theappearance of stereotypies. A dose of 25 μg ND-7000+25 μg XA led to avery marked potentiation of stereotypies (grooming) and decreased thelatency time to onset of these involuntary movements (2 minutes insteadof 5 minutes).

Study of ND-7000 on general activity of the animals in the open fieldtest

The test was performed 30 minutes after injection of the product at adose of 100 or 200 mg/kg per os. The observation period was 15 minutes.

At both doses tested, global activity of the animals decreased inproportion to the dose.

The results are presented in the following table:

Behavior Controls 100 mg/kg (i.p.) 200 mg/kg (i.p.) Grooming  5 ± 4  3 ±2  4 ± 2 Standing  53 ± 12  30 ± 6 (−44%)  20 ± 9 (−63%) Total squares215 ± 24 146 ± 15 (−33%) 106 ± 22 (−51%) Outer squares 188 ± 35 129 ± 11(−32%)  97 ± 23 (−49%) Inner squares  27 ± 13  17 ± 8 (−38%)  9 ± 7(−68%) The results are given as the mean ± SD (n = 5 rats/group)

When administered at a dose of 100 to 200 mg/kg per os, ND-7000 did notinduce catalepsy and did not alter reflex activities measured insensorimotor tests.

The actograph obtained after administration of ND-7000 100 mg/kg per os(p.o.) is shown in FIG. 38.

In conclusion, the effects of ND-7000 on cerebral dopaminergicactivities induced by XA and on global reactivity as measured in theopen field test, suggest that this product potentiates XA binding to itsreceptor. These results confirm the binding studies showing a markedincrease in XA binding to its receptor site in the presence of ND-7000.

These experiments provide pharmacological data confirming the existenceof another pathway by which to potentiate the effects of XA: syntheticligands which bind to the allosteric site. Negative allostericeffectors, antagonist of the binding of ND-7000 and kynurenic acidderivatives, which would reduce XA activity by decreasing its binding toits receptor site, might also be envisioned. This represents a novelavenue of research for the synthesis of original ligands.

13. An Example of an XA Receptor Agonist Ligand, a Xanthurenic AcidDerivative Named ND-1301

ND-1301 was chosen to illustrate the series of XA receptor agonistligands. This substance displaced radiolabelled XA from its binding sitewith good affinity and induced a strong electrophysiological response inpatch-clamp tests in NCB-20 cells. Oral administration of ND-1301 toanimals also modulated dopamine levels in brain tissue, as shown by theresults hereinbelow.

FIG. 39 illustrates the dose-response effect observed after p.o.administration of ND-1301 on dopamine tissue levels in different brainregions.

1. A method for selecting, identifying or characterizing compounds thatinhibit the binding of xanthurenic acid (XA) to proteins that bind XA,comprising: contacting a test compound with brain cell membranescontaining proteins which specifically bind XA, wherein said membranesare obtainable by the process comprising the following steps: (a)preparing synaptosomes by brain cells homogenisation to produce ahomogenate, and (b) recovering said brain cell membranes from saidhomogenate; and measuring the inhibition of the binding of xanthurenicacid to said proteins by said test compound, said measuring comprising(1) determining the binding of XA to the brain cell membrane proteins bycontacting a labeled XA with brain cell membrane containing saidproteins, (2) determining the binding of labeled XA to brain cellmembrane proteins in the presence of a test compound, and (3) comparingthe binding in (1) with the binding in (2) as an indication of saidinhibition.
 2. A method according to claim 1, wherein step (a) comprisesthe following steps: lysis of a brain sample to produce a lysate, andcentrifugation of the lysate to produce a pellet comprisingsynaptosomes; and step (b) comprises the following steps: disruption ofthe synaptosomes to produce brain cell membranes, and separation of thebrain cell membranes.
 3. The method of claim 1 wherein step (a) furthercomprises lysis of a brain sample and centrifugation of the obtainedlysate to provide synaptosomes, and step (b) further comprisesdisruption of said synaptosomes to produce a brain cell membranecontaining composition and recovering said brain cell membranes fromsaid brain cell membrane containing composition.
 4. A method accordingto claim 3, wherein step (a) comprises the following steps: homogenizingsaid brain samples in a volume of a solution S equal to 10× the weightof said brain sample (S=0.32 M sucrose, 10 mM KH₂PO₄ pH 6.0, 1 mM EDTA)to produce a homogenate sample, centrifuging said homogenate sample at915 g at 4° C. for 10 minutes to produce a first pellet and a firstsupernatant, separating the first supernatant from said first pellet toproduce a separated first supernatant, and centrifuging said separatedfirst supernatant at 18,200 g at 4° C. for 20 minutes to produce asecond pellet and a second supernatant; and step (b) comprises thefollowing steps: separating the second supernatant and the second pelletto produce a separated second pellet, wherein said separated secondpellet comprises synaptosomes, rupturing said synaptosomes in saidseparated second pellet by admixing a volume of distilled water at 0° C.equal to 70× the weight of said separated second pellet and homogenizingfor 30 seconds, to produce disrupted synaptosomes in a homogenizedcomposition, recovering said disrupted synaptosomes from saidhomogenized composition by centrifuging said homogenized composition at51,000 g at 4° C. for 20 minutes and separating a pellet produced bysaid centrifuging, said pellet containing said disrupted synaptosomes,washing said pellet in 50 mM KH₂PO₄ buffer pH 6.0 at 0° C. to produce awashed pellet composition and centrifuging said washed pelletcomposition at 51,000 g at 4° C. for 20 minutes to produce a supernatantand said brain cell membrane containing composition in the form of apellet, and recovering said brain cell membrane containing compositionin the form of a pellet.
 5. A method according to claim 1, wherein saidbrain cell membranes are synaptic membranes.